Divito et al S&TR 114(2011) Efectos a largo plazo fertilización con N

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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Soil & Tillage Research 114 (2011) 117–126 Contents lists available at ScienceDirect Soil & Tillage Research journal homepage: www.elsevier.com/locate/still Long term nitrogen fertilization: Soil property changes in an Argentinean Pampas soil under no tillage Guillermo A. Divito a, Hernán R. Sainz Rozas a,b,c, Hernán E. Echeverrı́a a,b,*, Guillermo A. Studdert a, Nicolás Wyngaard a a b c Facultad de Ciencias Agrarias, UNMDP, C.C. 276, (7620) Balcarce, Argentina E.E.A. INTA, Balcarce, Buenos Aires, Argentina CONICET, Argentina A R T I C L E I N F O A B S T R A C T Article history: Received 1 December 2010 Received in revised form 1 March 2011 Accepted 10 April 2011 Available online 17 May 2011 The main objective of nitrogen (N) fertilization is to achieve high yields and/or to increase grain quality. However, nutrient application may affect soil processes and cycles. These could involve increases in crop residues return to soil, changes in soil organic matter dynamic, NO3-N content and pH decrease. The aim of this study was to determine the effect of N application on: crop residue input, soil organic carbon (SOC) and N (SON), their particulate (POC and PON) and mineral associated (AOC and AON) fractions, mineralizable N (anaerobic incubations, AN), and pH on a Molisoll of the southern Buenos Aires Province under no tillage (NT). A long-term crop rotation experiment has been conducted between 2001 and 2008 on a complex of Typic Argiudoll and Petrocalcic Paleudoll soils at Balcarce, Argentina (378450 S, 588180 W). Three N rates (N0, N1 and N2) were evaluated, with an average N input of 0, 57 and 105 kg ha1 year1, respectively. Crop sequence was integrated by maize (Zea mays L.), soybean (Glycine max (L.) Merr.) and wheat (Triticum aestivum L.)/soybean double crop. Soil sampling was done in 2008, previous to maize planting. Nitrogen fertilization increased carbon (C) return to soil during 2001–2008 (11.1 and 18.7% for N1 and N2 respect to N0) but no differences in SOC, SON, AOC, and AON were observed among N rates in 0–5 and 0–20 cm depth. It was only found more PON in N1 and a slight tendency to increased POC (3% and 13% for N1 and N2 respect to N0) in 0–5 cm depth. At the same time, NO3-N content in 0–60 cm depth was similar among N rates (89.6  8.4, 88.6  6.4, and 81.6  10.3 kg N ha1 for N0, N1, and N2, respectively). By contrast, it was determined soil acidification (5.8  0.3, 5.5  0.2, and 5.3  0.2 for N0, N1, and N2, respectively) and AN reductions in 0–5 cm depth as N rate increased, (76.1  3.2; 74.9  6.3 and 57.9  3.5 for N0, N1 and N2 respectively). The high frequency of soybean in the rotation could have prevented higher increases in C return to soil and, as a consequence, mitigated the changes in related soil properties. In addition, the absence of N application to soybean also could have prevented enhances in soil acidification and AN depletion. ß 2011 Elsevier B.V. All rights reserved. Keywords: Nitrogen fertilization Long-term effect Soil organic matter No-tillage 1. Introduction The main objective of crop fertilization is to achieve high yields and/or increased grain quality (Mosier et al., 2009). However, nutrient application may affect soil processes and cycles (Russell et al., 2006). Nitrogen (N) is the main nutrient limiting crop growth and yield in the world (Huber and Thompson, 2007) and in the southeastern Buenos Aires Province (Argentina) in particular (Echeverrı́a and Sainz Rozas, 2005). Crop response to N fertilization * Corresponding author at: C.C. 276, (7620) Balcarce, Argentina. Tel.: +54 2266 439100x777; fax: +54 2266 439101. E-mail addresses: hecheverr@balcarce.inta.gov.ar, gastudde@gmail.com (H.E. Echeverrı́a). 0167-1987/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.still.2011.04.005 involves increases in CO2 fixation and, as a consequence, in aboveground and root biomass production (Tognetti et al., 2005). This increase leads to more crop residue return to soil (Studdert and Echeverrı́a, 2000; Wilts et al., 2004) which, in temperate agroecosystems, is considered the main factor controlling soil organic matter (SOM) dynamics (Stevenson and Cole, 1999). Therefore, several reports exist about rates and quantity of SOM change due to N fertilization, depending on N rates, crop rotation, environment and soil type evaluated (Álvarez, 2005). It is widely assumed that agriculture, particularly under conventional tillage (CT) leads to depletions of SOM content. Therefore, N fertilization is often recommended to increase SOM, or to reduce loss rates (Studdert and Echeverrı́a, 2000). Liebig et al. (2002) reported that fertilization with 180 and 90 kg N ha1 increased soil carbon (C) sequestration in 1.4 Mg ha1 and Author's personal copy 118 G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 1.0 Mg ha1, respectively, in comparison with the unfertilized plot. Nevertheless, both treatments showed SOM depletion in 0–7.6 cm depth respect to the level determined 26 years before. In the southeastern Buenos Aires Province, Fabrizzi et al. (2003) observed that in a degraded soil, N fertilization increased SOM content with respect to the unfertilized treatment, whereas the same treatment applied to a non-degraded soil did not cause differences. Contrarily, Khan et al. (2007) documented opposite trends for a wide variety of geographic regions, cropping systems, and tillage practices: mineral N application, at higher rates than those used currently in Argentina, enhanced microbial decomposition of SOM and reduced soil organic carbon (SOC) content in relation to the unfertilized plots. Most agricultural soils in the world and particularly in Argentina have net negative charge, which limits NO3 adsorption and increased leaching probability (McNeill and Unkovich, 2007). As a consequence, crop N uptake and microbial immobilization is considered the main N retention process in soils (Halvorson et al., 2002; Eriksen, 2008). Therefore, Halvorson et al. (1999) observed a positive relationship between the amount of N in residues and total N in 7.5 cm top-soil after applying N to crops during 10 years. However, as Khan et al. (2007) reported for SOC, Mulvaney et al. (2009) observed that N fertilization caused soil organic N (SON) depletions due to the increase in microbial activity. Changes in SON caused by N fertilization may affect soil N potential mineralization (N0), which plays a critical role in N supply to crops. This parameter can be estimated by the amount of N-NH4+ produced by short term anaerobic incubations (AN), which has been demonstrated to be sensitive to the effect of tillage, liming, fertilization and crop sequences (Soon et al., 2007). In the southeastern Buenos Aires Province, Diovisalvi et al. (2008) reported that after 10 years, crop N fertilization caused no change in SON in 0–5 cm and 0–20 cm depths. As a consequence, it was observed no difference in AN. On the contrary, Domı́nguez et al. (2009) and Genovese et al. (2009) reported AN depletions after seven and twelve years of N fertilization in the same region, respectively. Soil acidification is a negative aspect of applying ammonium N forms. This process can produce detrimental conditions for microorganism development, altering C and nutrient cycling (Kemmitt et al., 2006). Soil acidity also affects crops, reducing their biomass production and grain yield (Zhao et al., 2010). Soil organic matter dynamics is highly modified by tillage systems (Six et al., 1999). No-tillage (NT) generally leads to an increase of SOM content compared with CT that is more evident in the upper layers of the profile (Kern and Johnson, 1993; West and Post, 2002). As a consequence, some authors reported that SOM changes due to N fertilization were more pronounced in the first layer of soil (Fabrizzi et al., 2003; Domı́nguez et al., 2009). Acidification of soil surface has also been more intense in NT than in CT when N was broadcast-applied (Eckert, 1985). Traditionally, agricultural systems in the southeastern Buenos Aires Province (Argentina) comprised aggressive tillage that reduced SOM levels. Nowadays, most of these systems have adopted NT to mitigate soil degradation. In spite of that, soybean cultivation has dramatically increased, and this contributed to increase SOM losses (Studdert and Echeverrı́a, 2000). In this context, best management practices that involve adequate nutrient applications are auspicious to maintain or improve soil quality. The aims of this study were to evaluate the long term effect of N fertilization on crop biomass production, and some selected soil properties (BD, pH, AN, and SOC, SON, and their fractions) of a Mollisol of the southern Buenos Aires Province (Argentina) under NT. We hypothesized that long-term N crop fertilization (1) increases POC and PON and AN, (2) reduces pH and (3) does not affect BD and NO3-N content. 2. Materials and methods 2.1. Experimental site A long-term crop rotation experiment has been conducted between 2001 and 2008 at the Unidad Integrada Balcarce, at Balcarce, Buenos Aires Province, Argentina (378450 S, 588180 W, 138 m above sea level). The soil of the experimental site is a complex of Mar del Plata series (fine, mixed, thermic Typic Argiudoll) and Balcarce series (fine, mixed, thermic Petrocalcic Paleudoll) with less than 2% slope (no erosion). The petrocalcic horizon of the Balcarce soil series is below 0.7 m. The soil has a loam texture at the surface layer (0– 20 cm depth), with an average size-particle distribution of 23% clay, 36% silt and 41% sand. The subsurface layer (25–110 cm depth) has clay-loam texture. Prior to the establishment of the experiment, the site had been under cropping with CT for more than 25 years. Tillage comprised moldboard plowing, disking and field cultivation with the least tillage operations necessary to get an appropriate seedbed. In 2001, at the beginning of the study, soil pH in the 0–20 cm depth was 5.5 and SOC and P-Bray were 25.4 g kg1 and 28.7 mg kg1, respectively. 2.2. Experiment design In the southern Buenos Aires Province, the typical rotation cycle is maize (Zea mays L.), soybean (Glycine max (L.) Merr.) and wheat (Triticum aestivum L.)/soybean double crop. For maize and soybean, the optimal sowing dates are during October and November respectively, and the harvest period starts during April–May for both crops. Wheat is planted in June–July and harvested at the end of December or early January. Double cropped soybean is sown immediately after wheat harvest and is harvested during May. Crop sequence during the first rotation cycle was: maize (sown in year 2001), soybean (2002) and wheat/soybean double crop (2003); the second cycle was integrated by wheat/soybean (2004), maize (2005) and soybean (2006). In 2007 wheat/soybean double crop was sown, corresponding to the third rotation cycle. Three different N rate treatments (N0, N1, and N2), applied to cereal crops, were evaluated. Treatment N1 (70 kg N ha1) was defined in correspondence to the most usual rates applied for farmers in the region to wheat and maize and was. Treatment N2 (140 kg N ha1) was established in order to achieve maximum yields. In the maize sown in 2005 the N rates increased (116 kg N ha1 and 176 kg N ha1 for N1 and N2 respectively). In addition, N0 was used as a check, without N fertilization. Urea (46– 0–0) was the N source. Soybean was inoculated with Bradyrhizobium japonicum before sowing. The experimental design was in randomized complete blocks with four repetitions. The dimensions of the experimental units were 12  5 m. Phosphorus (P) and sulfur (S) were applied to all treatments as triple superphosphate (0–46–0) and gypsum (SO4Ca  2H2O, 16% S, 20% Ca), respectively. High potential varieties and hybrids, widely spread in the southeastern Buenos Aires Province, were used. Crops were sown under NT, in optimal dates for the region and were kept free of weeds, pests and diseases. The numbers of plants established per m2 were 8, 25, 300 and 35 for maize, soybean, wheat and double cropped soybean. Nitrogen was broadcast-applied at Zadoks 22 (Zadoks et al., 1974) in wheat, and V6 (Ritchie and Hanway, 1982) in maize. Phosphorus and S were also surface-broadcasted annually, previous to crop sown, applying 20 kg P ha1 year1 and 15 kg S ha1 year1. At physiological maturity, defined in Zadoks 90 (Zadoks et al., 1974) for wheat, R6 (Ritchie and Hanway, 1982) for maize, and R8 (Fehr and Caviness, 1977) for soybean, crops were harvested by collecting material from a surface of 8 m2 per plot. Crops residues were returned to soil. Author's personal copy 119 G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 The evapotranspiration (CET) of the double-cropped soybean (2008) was determined as the product between potential evapotranspiration (ET0) and crop coefficient (Kc). The ET0 was calculated according to Penman (1948) and the Kc (CET/ET0) values were those reported for the region by Della Maggiora et al. (2000). 2.3. C input estimation Aerial biomass residues were estimated considering crop yields and their harvest indexes (0.45, 0.40, and 0.45 for maize, soybean and wheat, respectively) (Carcova et al., 2004). Root C contribution and root exudates were calculated according to the relations root:aerial biomass production proposed by Buyanovsky and Wagner (1997) which were 0.35, 0.48, and 0.38 for maize, wheat and soybean, respectively. The proportion of the total contribution of root C and root exudate C located in the 0–20 cm depth was considered as 0.91, 0.90, and 0.84 for maize, wheat and soybean, respectively (Buyanovsky and Wagner, 1986). Carbon content of plant tissue was assumed as 43% (Sánchez et al., 1996). Wheat sown in 2007 suffered frost damage at flowering. In order to calculate its C return to soil it was assumed that the aboveground biomass production for all treatments corresponds to a grain yield of 4000 kg ha1. This assumption was based on the general crop condition at this stage of development, determined by visual estimation. Nitrate N content was determined by steam microdistillation (Bremner and Keeney, 1966). For AN determination, 10 g of soil (only 0–5 and 5–20 cm layers) were incubated under anaerobic conditions at 40 8C for 7 d. The NH4+-N produced during the incubation was determined by steam microdistillation (Bremner and Keeney, 1966), according to the method proposed by Gianello and Bremner (1986). Soil samples from 0 to 5 and 5 to 20 cm were analyzed for SOC contents by wet combustion with maintenance of the oxidation reaction temperature (120 8C) for 90 min (Schlichting et al., 1995). Soil organic N content was determined in the same layers by dry combustion and N thermo-conductivity detection (LECO, 2010). Mineral-associated organic matter was separated by dispersing soil aggregates with hexametaphosphate and passing the dispersed samples through a 53 mm sieve (Cambardella and Elliott, 1992). Soil slurry that passed through the sieve contained the mineral-associated C and N (AOC and AON, respectively). Water in the slurry was evaporated in a forced air oven at 45 8C and the dried sample was ground with a mortar and pestle and analyzed for organic C (AOC) and total N (AON) contents as described above. Particulate organic C and N (POC and PON) contents were calculated by subtracting AOC and AON from SOC and SON, respectively. Results of both organic C and organic N fractions were expressed as concentration (g kg1). Carbon:nitrogen ratio (C:N) was calculated for the whole soil and for each fraction. Carbon sequestration efficiency (CSE) was calculated as: 2.4. Cumulative N variation Annual N variation was calculated as the difference between fertilizer N applied (kg ha1) and N crop extraction in grain (kg ha1). Grain N concentration was determined by dry combustion at 950 8C and N thermo-conductivity detection with a TruSpec CN analyzer (LECO, 2010). For wheat/soybean double crop, there were considered both crop N extractions. For soybean N balance, it was assumed that 0.44 of N was fixed from the air (Collino et al., 2009). Cumulative N variation was estimated as the sum of each year variation. CSE ¼ ðSOC 2008  SOC 2001Þ=C i where SOC 2001 and SOC 2008 are the SOC (kg ha1) in 0–20 depth in each year and Ci is the carbon input (aboveground residue biomass, root biomass, and root exudate C) in the same depth during the period 2001–2008. The BD considered to estimate SOC pool in 0– 20 cm depth was 1.27 Mg m3. This value was similar to the observed by Domı́nguez, G.F. (pers. comm.) in a similar long-term experiment placed next to this one and also coincided with the BD determined in 2008 for 0–10 cm depth (on average for the three N rates and 0–5 cm and 5–10 cm). 2.5. Soil sampling and laboratory procedures 2.6. Statistical analyses Composite soil samples (5 sub-samples per plot) were taken in 2008, shortly before maize planting, from 0 to 5, 5 to 20, 20 to 40 and 40 to 60 cm depth layers. Soil samples were dried at 30 8C and ground to pass a 2 mm sieve. Recognizable crop residues and roots retained on the 2 mm sieve were eliminated. Soil bulk density (BD) was measured at the same date, using a core sampler of 50.0 mm diameter and 50.0 mm height. Undisturbed soil cores (4 sub-samples per plot) were taken randomly from each treatment at 0–5 and 5–10 cm depths. The soil around the cylinder was excavated and the cylinder with the core in place was removed by sliding a shovel below the cylinder. Cores were dried at 105 8C and weighed. Bulk density was calculated by dividing the core mass by their volume. Soil pH was determined at a 1:2.5 (w:w) soil-to-water ratio. Treatment effects were evaluated by analysis of variance using SAS (the Statistical Analysis System, SAS Institute, 1985) and all the effects were treated as fixed (PROC GLM, Littell et al., 1991). When Fstatistic for treatments was significant, Least Significant Difference (LSD) at the 0.05 level was calculated. Linear relationships between soil properties were tested using PROC REG (SAS Institute, 1985). 3. Results and discussion 3.1. Crop C input Wheat and maize increased their C input to soil in response to N fertilization (P < 0.05, Table 1). On the average, the response of Table 1 Carbon input (aboveground biomass residues, roots and roots exudates) from N fertilized crops (N0, N1 and N2) during 2001–2008. kg ha1 N0 N1 N2 Maize 2001 Soybean 2002 Wheat 2003 Soybean 2004 Wheat 2004 Soybean 2005 Maize 2005 Soybean 2006 Wheat* 2007 Soybean 2008 Sum 5783 b 7741 a 8244 a 2723 a 2545 a 2647 a 5879 b 7590 b 9620 a 2096 a 696 b 669 b 3695 b 6078 a 6215 a 1531 a 863 b 785 b 7813 b 8818 ab 9091 a 3314 a 3206 a 3155 a 5553 5553 5553 1606 a 1378 a 1492 a 39993 c 44468 b 47471 a For each year, similar letters indicates statistically equal values among N rates at P < 0.05 using Fisher protected LSD. * Carbon return to soil correspond to a grain yield of 4000 kg ha-1. Author's personal copy G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 3.2. Soil bulk density Soil BD did not differ among N rates in 0–5 (P = 0.7, 1.27  0.07; 1.25  0.04 and 1.27  0.04 Mg m3 for N0, N1 and N2 respectively) and 5–10 cm depth (P = 0.99, 1.28  0.04; 1.28  0.01 and 1.28  0.04 Mg m3 for N0, N1 and N2 respectively). These observations are in agreement with the findings reported by Domı́nguez et al. (2009) for the same region, who determined no differences in BD in the 0–5 and 5–20 cm layers due to N application during seven years. Haynes and Naidu (1998) stated that the increase in SOM inputs and the stimulation of soil microbial and faunal activity due to inorganic fertilizations could improve soil aggregation, increase soil porosity and, as a consequence, reduce BD. Therefore, since no differences among treatments were determined in SOC (Fig. 1), it was consistent that the BD resulted similar among N rates. On the contrary, Halvorson et al. (2002) reported that BD decreased from 1.45 to 1.40 Mg m3 as the N rate increased from 0 to 46 kg N year1 applied to wheat in a monoculture in a 23-year long term study under NT. 40 POC AOC 0 to 5 cm SOC (g kg-1) 35 30 25 20 15 10 5 0 0 1 2 N rate 40 5 to 20 cm 35 SOC (g kg-1) cereals was 28.2% to N1 and 39.0% to N2. First-season soybean sown on plots previously fertilized with different N rates did not differ in yield among treatments. However, double-cropped soybean yield in 2004 and 2005 growing seasons was reduced as N rates applied to wheat increased (P < 0.01, Table 1). In the southeastern Buenos Aires province, it was determined that Nfertilized wheat increased water evapotranspiration up to 105 mm during the growing season (Caviglia and Sadras, 2001). This has negative implications in water availability for the double-cropped soybean. In addition, a common problem observed on field-scale crops are failures in soybean sowing (i.e. high variability in stand uniformity) caused by the amount of aboveground wheat residues. This problem was more important in high-yield wheat (N fertilized), and could have been responsible of the reductions in double cropped soybean plant population and yield observed in this experiment. Carbon input increase due to N fertilization indicates that soil N supply was lower than crop N demand. For the region, Sainz Rozas et al. (2008) and Barbieri et al. (2009) have reported high N responses of cereal crops, especially under NT where SOM mineralization is low. However, these N responses were smaller than observed in other crop rotations or soils. For continuous maize, Jagadamma et al. (2008) determined average increases of 97.7% in annual aboveground residues inputs. In addition, Varvel (2000) reported that the effect of N fertilization on normalized grain maize yields was consistent in all crop rotations, but it was greater in continuous maize (N responses of ca. 95% for continuous maize and ca. 15% for maize/soybean). Results of the first season soybean coincide with those reported by Jagadamma et al. (2008), who determined that soybean yield was not affected by N applied to the preceding corn crop. This could be a consequence of its capability to regulate atmospheric N fixation in response to soil inorganic N content (Salvagiotti et al., 2008). Crop rotations including maize, soybean and wheat/soybean double crop are the most typical in the southeastern Buenos Aires Province. Given soybean capability to fix atmospheric N, the effect of N fertilization was only evaluated in both cereals. This aspect should be specially considered, because it is fully accepted that the crops included in a rotation are more important to promote changes in soil properties than fertilization management (Liebig et al., 2002; Russell et al., 2006). Jagadamma et al. (2007) reported that under continuous maize, the additional mass of residues returned to soil produced by N fertilization was higher than that determined under maize/soybean rotation. As a consequence, the high frequency of soybean in the rotation of this experiment may have prevented higher increases in C return to soil as a consequence of N fertilization. 30 25 20 15 10 5 0 0 1 2 N rate 0 to 20 cm 40 35 SOC (g kg-1) 120 30 25 20 15 10 5 0 0 1 2 N rate Fig. 1. Soil organic carbon (SOC), associated organic carbon (AOC) and particulate organic carbon (POC), expressed as concentration in the 0–5 cm (upper graph), 5–20 (central graph) and 0–20 cm (bottom graph) layers for three N rates (N0, N1 and N2). Vertical bars for each column portion indicate standard error of the mean. 3.3. Soil organic carbon Despite the increases observed in C input (Table 1), N fertilization did not produce differences in SOC and its fractions after seven years in 0–5 cm depth (P = 0.21, P = 0.69 and P = 0.38 for SOC, AOC and POC respectively). In addition, in 5–20 and 0–20 cm depth the concentration of SOC and its fractions were also similar among treatments (P = 0.96, P = 0.84 and P = 0.50 for SOC, AOC and POC in 5–20 and P = 0.81, P = 0.92 and P = 0.34 for SOC, AOC and POC in 0 to 20 respectively) (Fig. 1). Particulate organic C is a labile fraction and can respond rapidly to changes in residue inputs (Janzen et al., 1998; Galantini and Rosell, 2006). However, it only showed a tendency to increase in the 0–5 cm depth (3% and 13% for N1 and N2 respect to N0). These results support the importance of N fertilization for increasing residue return, but demonstrate that it is not sufficient to increase SOC level in these soils. Diovisalvi et al. (2008) and Domı́nguez et al. (2009) reported similar results in an experiment next to this study, but others, with other crop rotations, tillage systems and periods under evaluation, reported different trends. For example, for similar soils Studdert and Echeverrı́a (2000) observed that eleven years of CT decreased SOC concentration by 4.1–8.8 g kg1 without supplemental N and by 2.8–7.2 g kg1 when N fertilizer was applied. In addition, Fabrizzi et al. (2003) reported contrasting responses in SOC change due to N fertilization. In undegraded soils under a crop rotation including pastures, they observed that four-year fertilization with Author's personal copy 121 G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 CSE ( kg-1 kg-1) 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 N rate Fig. 2. Carbon sequestration efficiency (CSE, kg kg1) during the 2001–2008 period for three N rates (N0, N1 and N2). Vertical bars for each column indicate standard error of the mean. 150 kg N ha1 year1 did not generate SOC differences comparing to unfertilized plots. In contrast, in a degraded soil with more than 25 years of cropping under CT, N fertilization with 120 kg N ha1 year1 during seven years increased SOC in the 0– 7.5 cm depth. This suggests that N fertilization is a significant strategy in order to correct soil degradation due to intensive cropping, but in situations like those of this experiment (i.e. high SOM contents, NT, and high C return as crop residues), the benefits could not be evident. Averaging the three N rates, SOC content in the 0–20 cm showed an increase with respect to 2001 (28.4 and 25.4 g kg1 in 2008 and 2001, respectively) which indicates that during this period C input was greater than heterotrophic C respiration outputs. These results explain the positive values of CSE (Fig. 2) and are relevant because soils in the region have shown SOC content decreases as a consequence of cropping intensification without rotating with pastures (Studdert et al., 1997). For this region, Studdert and Echeverrı́a (2000) reported higher SOC depletion rates as soybean increases its frequency in one crop per year rotations under CT. On the contrary, in the present study the high frequency of soybean did not produce SOC depletions under NT. In this case, the presence of double cropped soybean should be considered as positive, due to the additional C input to soils (3705 kg C ha1 on average for the N rates during the experiment period, Table 1). This agrees with Álvarez and Steinbach (2008) who estimated that wheat/soybean double crop was the crop sequence that caused the least SOC depletion rates in the north of Maize Soybean 2001 2002 the Argentinean Pampas because of the additional C input by double cropped soybean. No differences in CSE were determined among N rates (P = 0.88). On the contrary, some researchers reported that high N rates applications enhanced microbial activity and CO2 loses (Russell et al., 2005) and, as a consequence, reduced CSE. Khan et al. (2007) reported that N fertilization during 40 years caused increases in microbial activity and, therefore, SOC reductions in the 0–40 cm depth, despite crop residue C input was greater. Similarly, in the southeastern Buenos Aires Province, Domı́nguez et al. (2009) suggested that N fertilization speeded up C turnover because the greater C input during 7 years did not lead to SOC content increase. Our results would indicate that N fertilization did not raise CO2 outputs because a similar proportion of returned C was retained as SOC. This could be a consequence of the relative low N rates applied, in comparison with those reported by Khan et al. (2007). 3.4. Organic nitrogen Cumulative soil N variation was negative for the three N rates and was more pronounced as the N rate decreased (Fig. 3). For N0 and N1 annual N variation was negative all years (40.1 and 17.2 kg N ha1 year1 respectively) while for N2 the variation was positive when N was applied to cereals (Fig. 3) (except in wheat/soybean sown in 2003). These results put in evidence the negative effect of soybean on soil N balance despite its capability to fix atmospheric N. Although the difference between fertilizer N inputs and grain N outputs is a simplified consideration of soil gain and losses (Meisinger et al., 1992), it gives an approach of soil N accumulation or depletion. Therefore, it demonstrates that none of the treatments could have increased soil N level, as N outputs in grain were greater than inputs. There were no differences among N rates in SON (P = 0.07) and AON (P = 0.66) in 0–5 cm depths although PON concentration was higher (P < 0.05) in the treatment that received intermediate N rate during seven years (N1). In 5–20 and 0–20 cm depths there were no differences among N rates in SON and their fractions (P = 0.88, P = 0.65, and P = 0.94 for SON, PON, and AON, respectively, in 5–20 cm and P = 0.93, P = 0.67, and P = 0.99 for SON, PON and AON, respectively, in 0–20 cm depth) (Fig. 4). In general, these results agree with Domı́nguez et al. (2009) and Diovisalvi et al. (2008) observations in the same region. They reported similar SON, Wheat/Soybean Wheat/Soybean 2003 Maize Soybean Wheat/Soybean 2005 2006 2007 2004 -1 Cumulative N balance (kg ha ) 200 a 0 b a b c a a b c -200 a b a a b c b b c -400 N0 N1 N2 c c c -600 Fig. 3. Cumulative nitrogen mass balance for the three N rates (N0, N1 and N2) in the 2001–2007 period. Vertical bars for each point indicate standard error of the mean. For each year, similar letters indicates statistically equal values among N rates at P < 0.05 using Fisher protected LSD. Author's personal copy 122 G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 a b 0 to 5 cm 0 to 5 cm 100 a b -1 3 PON p< 0.05 AN (mg kg ) SON (g kg-1) 4 2 1 b 80 60 40 20 0 0 0 N rate PON 3 100 -1 2 1 2 5 to 20 cm 80 a 60 a a 40 20 0 0 0 N rate 4 1 2 N rate 100 0 to 20 cm -1 AN (mg kg ) SON (g kg-1) 1 N rate 5 to 20 cm AON AN (mg kg ) SON (g kg-1) 4 ab 3 2 0 to 20 cm 80 60 a a a 40 20 1 0 0 N rate 0 1 2 N rate Fig. 4. Soil organic nitrogen (SON), associated organic nitrogen (AON) and particulate organic nitrogen (PON), expressed as concentration in the 0–5 cm (upper graph), 5–20 (central graph) and 0–20 cm (bottom graph) layers for the three N rates (N0, N1 and N2). Vertical bars for each column portion indicate standard error of the mean. For PON fraction in 0–5 cm depth, similar letters on top of the column indicate statistically equal values at P < 0.05 using Fisher protected LSD. Fig. 5. Anaerobic nitrogen (AN, mg kg1) in 0–5 cm (upper graph), 5–20 (central graph) and 0–20 cm (bottom graph) layers for the three N rates (N0, N1 and N2). Vertical bars for each column indicate standard error of the mean. Similar letters on top of the column of each figure indicate statistically equal values at P < 0.05 using Fisher protected LSD. AON and PON in N fertilized and unfertilized soil. Conversely, Jagadamma et al. (2008) reported that after 23 years, SON concentration increased as much as N rates applied to maize did. The higher PON concentration in N1 plots with respect to N0 agree with the tendency observed in POC (Fig. 1) and could be a consequence of the greater N input. Contrarily, PON difference between N1 and N2 did not agree with the tendency observed in POC for these treatments (Fig. 1). Moran et al. (2005) reported that in laboratory conditions N addition to soil and residues enhanced microbial activity and, as a consequence, increased soil N transformations to more recalcitrant forms. Nevertheless, observed NOP differences had little agronomic importance, considering the high SOM content of soils in the southeastern Buenos Aires Province. Soil C:N ratio did not differ among N rates in 0–5 cm depth (P = 0.08) and were 12.4  0.7, 11.7  0.7, and 12.4  0.3 for N0, N1, and N2, respectively. In 5–20 cm C:N was also similar (P = 0.72), being 11.6  0.4, 11.5  0.5, and 11.3  0.5 for N0, N1, and N2, respectively. These results are consistent with those reported by Domı́nguez et al. (2009) and Fabrizzi et al. (2003) for similar environmental conditions. Halvorson et al. (1999), working on an Argiudoll of the United States, also reported that 11 years of N fertilization produced no differences in C:N ratio, in 0–7.5 and 7.5–15 cm depths. Conversely, Jagadamma et al. (2008) reported that N fertilization during 23 years increase SOC and SON with reductions in C:N ratios (12.7 and 11.9 for unfertilized and fertilized treatments, respectively). These results could be partially explained by de higher N rates applied. Taking into account the differences determined in cumulative N balance (Fig. 3) and the absence of variation in SON, SON fractions, and C:N ratios among N rates, it can be inferred that N losses occurred in fertilized treatments. This issue will be discussed in Section 3.7. 3.5. Soil pH Soil pH in 0–5 cm depth was lower at higher N rates (P < 0.01, 5.8  0.3, 5.5  0.2, and 5.3  0.2 for N0, N1, and N2, respectively). In 5–20 cm depth there were no differences among treatments (P = 0.68, 5.8  0.4, 5.8  0.1, and 5.7  0.03 for N0, N1, and N2, respectively). These observations agree with Mahler and Harder (1984) who reported an intense acidification of soil surface (0–7.5 cm) under NT when N was broadcast. Similarly, Liebig et al. (2002) reported soil acidification in 0–7.5 cm and 0–30 cm after 16 years of N fertilization in an Argiudoll of Iowa. This decrease was less pronounced in a maize/ soybean rotation than in a maize monoculture, because soybean is not N fertilized. These results suggest that the presence of soybean in the rotation could help to mitigate soil acidification. In addition, S was Author's personal copy 123 G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 applied to all treatments as gypsum, which could increase soil pH due to the H+ replacement by Ca2+. Therefore, although soil acidification could have been attenuated by this mechanism, it was not considered as relevant because the low rate applied (83 kg gypsum ha1 year1). The main cause of soil acidification is the production of H+ due to urea hydrolysis. Each mole of N-urea oxidized to NO3 produces one mol of H+. On the other hand, NO3 uptake by plants leads to the release of an equivalent amount of OH into the rhizosphere, resulting in the neutralization of acidity. Contrarily, leaching of NO3 from the root zone causes permanent acidification by uncoupling the proton balancing system. As a consequence, and in agreement with the absence of differences observed in SON and their fractions, NO3 losses in N1 and N2 could be the cause of acidification in 0–5 cm. Nitrogen applications can also have indirect effects on soil acidification. This can be explained by the increase in cation removal caused by increased crop productivity. Therefore, the surplus of cations over anions in cereal grain is small in comparison with legumes (Pierre and Banwart, 1973). In this study, yield increases due to N fertilization occurred in maize and wheat, and soybean in the double crop showed an opposite tendency, so acidification due to excess cation removal in grain was estimated to be small. 3.6. Anaerobically mineralized nitrogen The application of the highest N rates (N2) generated less AN with respect to N0 and N1 in 0–5 cm depth (P < 0.05). However, in the 5–20 cm and 0–20 cm depths there were no differences among N rates (Fig. 5, P = 0.17 and P = 0.21, respectively). These results agree with those reported by Domı́nguez et al. (2009) and Genovese et al. (2009) for long-term experiments of 7 and 12 years, respectively, in the same region. Those authors indicated that in the 0–20 cm depth, the unfertilized treatments showed higher AN than the N fertilized ones. Similarly, Carpenter-Boggs et al. (2000), observed that N mineralization in an unfertilized Calcic Hapludoll soil was higher, although C and N input in residues was greater when N fertilizer had been applied to crops. Priming effect is frequently mentioned as the responsible of the decrease in N potential mineralization because N application enhances SON turnover and, as a consequence, reduces the mineralizable N pool (Kuzyakov et al., 2000). In this study there 0 to 5 cm 5 to 20 cm 80 -1 AN (mg kg ) -1 AN (mg kg ) 80 60 40 20 60 40 20 0 20 25 30 35 0 20 40 25 30 -1 35 SOC (mg kg-1) SOC (mg kg ) 0 to 5 cm 5 to 20 cm 80 -1 AN (mg kg ) -1 AN (mg kg ) 80 60 40 60 40 20 20 0 0 0 5 10 0 15 5 POC (mg kg-1) 10 15 -1 POC (mg kg ) 0 to 5 cm 5 to 20 cm 80 -1 -1 AN (mg kg ) 80 AN (mg kg ) 40 60 40 40 20 20 0 0.3 60 0.5 0.7 PON (mg kg-1) 0.9 1.1 0 0.3 0.5 0.7 0.9 1.1 -1 PON (mg kg ) Fig. 6. Relationships between anaerobically mineralized nitrogen (AN) and soil organic carbon (SOC, upper graph) particulate organic carbon (POC, center graph) and particulate organic nitrogen (PON, bottom graph) in 0–5 cm (left) and 5–20 cm (right). Author's personal copy 124 G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 3.7. Nitrate N Soil NO3-N content in the 0–60 cm depth did not differ among N rates (P = 0.41, 89.6  8.4, 88.6  6.4, and 81.6  10.3 kg N ha1 for N0, N1, and N2, respectively) in spite of the cumulative differences 0 to 5 cm AN (mg kg-1) 100 80 y = 30,5x - 98,6 R² = 0,51 60 40 20 0 5.0 5.2 5.4 5.6 5.8 6.0 pH 5 to 20 cm 100 80 AN (mg kg-1) was no evidence of this process, since no differences in CSE were determined (Fig. 2). This suggests that other processes could be involved in AN decrease with higher N rates. There was no association between AN and SOC, POC and PON in 0–5 cm and 5–20 cm depths (P > 0.05) (Fig. 6). In general, close associations between AN and SOC, SON, and their labile fractions were determined in different agricultural soils in the world (Westerhof et al., 1998) and in the southeastern Buenos Aires Province in particular (Fabrizzi et al., 2003). This relation could have been expected because SOM is a substrate for the heterotrophic microorganisms responsible for N mineralization. However, these determinations were done in soils with a wide range of managements, such as different tillage systems (Fabrizzi et al., 2003). Sharifi et al. (2007) reported that in soils of North America with a wide range of SOC and NOC contents, both properties were highly correlated with potentially mineralizable N (N0). Although, when each soil was considered individually no association was observed. Similarly, Sainz Rozas et al. (2008) determined that in 26 soils of the southeastern Buenos Aires Province under NT with a relatively narrow range of SOC contents (29–38 g kg1 in 0–20 cm depth) there was no significant relationship between it and N mineralization potential. Although a positive correlation between AN and pH was determined (P < 0.05) in the 0–5 cm depth, it was not observed in the 5–20 cm depth (Fig. 7). Soil organic C and SON cycles are mainly controlled by pH, because it affects microbial diversity, activity, and biomass (Paul, 2007). Nitrogen mineralization can occur in a wide range of pH’s but the optimum value is between 6.0 and 8.0 (Paul, 2007). Soon et al. (2007) reported that lime application to soil during 10 years increased soil pH (6.0 and 5.3 for limed and unlimed soils, respectively) and biological N mineralization indicators, as aerobic and anaerobic mineralizable N increased too. However, hot KCl-extractable N (Gianello and Bremner, 1986) did not differ between treatments, which demonstrate that either microbial biomass or activity were responsible for the reductions in N mineralization. Soon and Arshad (2005) suggested that it was the turnover of the increased microbial N what resulted in the higher mineralizable N associated with liming of acid soils. In agreement with this, Liebig et al. (2002) reported that soil acidification in the 0–30 cm depth after 16 years of N fertilization was directly associated with reductions in microbial biomass. Although it was demonstrated that soil pH affects mineralization in field conditions and in short aerobic incubations, its effect on short anaerobic incubations is not completely known. As a consequence, it is important to determinate if the reduction in NH4+ during the incubation is proportional to the reduction in mineralization on field conditions. This will be helpful to improve estimations of soil potential mineralization, especially because AN is widely used for crop N fertilization recommendations in the southeastern Buenos Aires Province (Sainz Rozas et al., 2008). 60 40 20 0 5.0 5.2 5.4 5.6 5.8 6.0 pH Fig. 7. Relationships between anaerobically mineralized nitrogen (AN) and soil pH in 0–5 cm (upper graph) and 5–20 cm (bottom graph). between N fertilizer inputs and N outputs in grain were higher as N rate decreased (Fig. 3). In addition, previous wheat/soybean double crop (2007) clearly showed that tendency in N mass balance (87.9  10.0, 13.0  4.8, and 54.0  8.3 kg N ha1 for N0, N1, and N2, respectively), so it could have been expected differences in NO3-N content at soil sampling in 2008. The obtained results could be a consequence of N losses during the crop growing period or along the period between crops. In the southeastern Buenos Aires Province, NO3-N leaching beyond soil rooting profile is the main cause of N losses (Echeverrı́a and Sainz Rozas, 2005). In this experiment, precipitations were higher than the CET during the double-cropped soybean grown in early 2008, showing a surplus of 197 mm in March (a few months prior to soil sampling and just at the beginning of the cool fall). In addition, in the period between crops (April to October of 2008, Table 2) rainfall could have also contributed to nitrate leaching, because soil profile was already saturated with water. As it was previously stated, N losses mechanisms may also explain the absence of the differences determined in SON and their fractions because they prevent the occurrence of N immobilization in organic forms. In this case, all the loss mechanisms that operated during the entire period under study could have contributed to reduce the differences among treatments in the N mass balance, due to the increase in N outputs in the fertilized plots. Therefore, besides the relevance of lixiviation in the region, NH3 volatilization is an important loss mechanism when urea is applied to maize. Sainz Rozas et al. (1999) reported that losses by this mechanism Table 2 Decadic and monthly rainfall for the period November 2007 to October 2008 and the historical monthly rainfall (1971–2006). 1st decade 2nd decade 3rd decade Sum 1971–2006 N D J F M A M J J A S O 16 28 5 49 94 26 0 0 26 83 61 4 2 156 111 47 36 65 148 90 210 28 2 240 102 0 14 2 16 77 0 0 17 17 56 11 21 7 40 54 29 21 26 75 46 10 7 54 71 56 1 0 18 19 58 14 5 10 28 96 Author's personal copy G.A. Divito et al. / Soil & Tillage Research 114 (2011) 117–126 could reach up to 11.4% of the urea-N broadcast in V6, and they determined that the proportion of N volatilized was higher as the N rate increased, mainly because the buffer capacity of the soil was saturated. In addition, Sainz Rozas et al. (2001) reported about 12 kg N ha1 losses by denitrification for the entire maize growth period when 210 kg N-urea ha1 had been broadcast at planting. In contrast, N losses by ammonium volatilization from the urea applied to wheat or by denitrification during the crop growing period are not considered as important. Ammonium volatilization is not significant in the region due to the low temperatures that occur when urea is applied to wheat (Videla et al., 1996). In addition, Picone et al. (1997) also determined low denitrification rates during early wheat stages in the same region, mainly due to the same temperature effect. 4. Summary and conclusions The results obtained show that N fertilization produced more C return to the soil during the period 2001–2008 but no differences in SOC, SON, AOC and AON were observed among N rates. In addition, contrary to the hypothesis, no differences in POC were determined and only a slight increase in PON with N1 was observed. As it was also stated, it was determined soil acidification (on 0–5 cm depth) as N rate increased and the NO3-N content and BD were similar through N rates. Besides, there were observed AN reductions as N rate increased, which did not agree with the hypothesis. It could be a consequence of the detrimental conditions to biological mineralization caused by soil acidification. The high frequency of soybean in the rotation could have prevented higher increases in C return to soil due to N fertilization, in comparison with sequences that included more cereal crops. As a consequence, it could have helped to mitigate the changes in the related soil properties such as SOC, SON, BD and AN. In addition, the lack of N application to soybean could also have attenuated the reductions in soil pH and in AN. Contrarily to the hypotheses of this work respect to the beneficial effects of N fertilization on soil properties, slight negative consequences were observed. Therefore, the evidence of soil surface acidification and AN reductions observed in this experiment should be considered, in order to prevent them, especially in crop rotations that receive higher N inputs or during longer periods of time. Acknowledgments We want to express our gratitude to J.L. Costa and his group for providing the facilities and material to determine soil physical properties. This study supported thanks to the financial support of the INTA Project AERN 295561, the FCA-UNMP AGR319/10 and FONCyT-PICT 2007-446. References Álvarez, R., 2005. A review of nitrogen fertilizer and conservation tillage effects on soil organic carbon storage. Soil Use Manage. 21, 38–52. Álvarez, R., Steinbach, H.S., 2008. Balance de carbono en suelos cultivados. In: Álvarez, R. (Ed.), Materia orgánica. Valor agronómico y dinámica en suelos pampeanos. Editorial Facultad de Agronomı́a, Universidad de Buenos Aires, Buenos Aires, Argentina, pp. 41–68. 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