Most of the world s rice is grown in fields surrounded by

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1 Soil Carbon and Nitrogen Changes in Long-Term Continuous Lowland Rice Cropping SOIL FERTILITY & PLANT NUTRITION Mirasol F. Pampolino Eufrocino V. Laureles Crop and Environmental Sciences Division International Rice Research Institute (IRRI) DAPO Box 7777 Metro Manila Philippines Hermenegildo C. Gines Philippine Rice Research Institute Science City of Muñoz 3119 Nueva Ecija Philippines Roland J. Buresh* Crop and Environmental Sciences Division International Rice Research Institute (IRRI) DAPO Box 7777 Metro Manila Philippines Rice (Oryza sativa L.), the main staple food in Asia, is typically produced on submerged anaerobic soils, which generally have slower decomposition of soil organic matter (SOM) than aerobic soils. We sampled four long-term experiments in the Philippines, with two or three rice crops grown each year with continuous or near-continuous soil submergence, to determine the effect of fertilizer management on long-term changes in soil C and N and on C and N balances. Soils were an Aquandic Epiaquoll, an Entic Pellustert, and a Typic Pelludert; soil ph ranged from 5.9 to 6.7. After 17 to 21 yr of continuous rice cultivation, the concentration of total soil organic C (SOC) and total soil N (N T ) in the topsoil (0 20 cm) were greater with N P K fertilization than without fertilization. During 15 yr of additional continuous rice cropping, topsoil SOC and N T were consistently maintained or increased regardless of N P K fertilizer regime. Topsoil SOC increased up to 10% in an experiment with three rice crops per year and removal of all aboveground plant biomass after each crop. Subsoil SOC and N T (20 80 cm) were not affected by fertilization. The N balances indicated that biological N 2 fixation averaged 19 to 44 kg N ha 1 crop 1 across the four experiments. Anaerobic N mineralization (ANM) in the topsoil was maintained during 15 yr of continuous rice cropping with N P K fertilization in all four experiments. The results suggest that continuous cultivation of irrigated rice with balanced fertilization on submerged soils maintained or slightly increased SOM and maintained soil N-supplying capacity. Abbreviations: ANM, anaerobic N mineralization; BIARC, Bicol Integrated Agricultural Research Center; BNF, biological dinitrogen fixation; LTCCE, long-term continuous cropping experiment; LTFE, long-term fertility experiment; N T, total soil nitrogen; PhilRice, Philippine Rice Research Institute; POC, permanganate-oxidizable carbon; SOC, soil organic carbon; SOM, soil organic matter. Most of the world s rice is grown in fields surrounded by earthen levees to retain rain and irrigation water and ensure soil submergence during at least part of the rice-cropping period. This submergence results in the depletion of soil O 2 and proliferation of anaerobic soil microorganisms. The decomposition of plant residues and organic matter is typically slower in submerged than in aerated soil (Acharya, 1935) and prolonged soil submergence can favor the maintenance or increase of SOM (Cassman et al., 1995; Bronson et al., 1997; Witt et al., 2000). Rice fields, however, often dry in the interval between rice crops, and rice fields sometimes even dry during portions of the rice-cropping period. This drying can lead to aerobic soil conditions, which favor faster decomposition rates. Frequent cycling between soil drying and wetting can stimulate microbial activity and enhance the rate of CO 2 evolution and organic matter decomposition (Sahrawat, 2004). The intensity of rice cropping i.e., the number of rice crops per year influences the duration of soil anaerobic and Soil Sci. Soc. Am. J. 72: doi: /sssaj Received 20 Sept *Corresponding author (r.buresh@cgiar.org). Soil Science Society of America 677 S. Segoe Rd. Madison WI USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. aerobic periods, which in turn could affect the dynamics of SOM. Increased rice-cropping intensity and the associated increase in soil submergence can lead to increased production of aquatic biomass mainly algae and an increase in incorporated crop residues, which decompose relatively slower than in aerated soils (Powlson and Olk, 2000). Intensive cultivation of two and three irrigated rice crops per year with the corresponding submergence of soil can promote a buildup of less-decomposed substances, which become incorporated into young SOM fractions. This has been associated with a reduced N-supplying capacity of rice soils (Olk et al., 1996; 2000). Long-term experiments are vital for examining the sustainability of cropping or land management systems (Greenland, 1994), and they enable the direct quantification of gradual changes in SOM resulting from modified management practices (Powlson and Olk, 2000). Previous studies have shown that the long-term application of inorganic fertilizers can maintain or increase SOM compared with no fertilizer application in experiments involving one or two rice crops per year (Yadav et al., 1998; Manna et al., 2005). Long-term rice cultivation may also increase SOM in the subsoil, as reported in China (Zhang and He, 2004). We conducted this study to determine the effect of fertilizer management on (i) changes in SOC and N T and (ii) C and N balances for rice soil systems during a 15-yr period of continuous cultivation with two or three crops of irrigated rice per year. Soil C and N were determined through concurrent analyses of archived soil samples collected at the start and finish of the time period. We also investigated SOC and N T in the soil profile as affected by fertilization after 32 to 37 yr of continuous rice cropping. 798 SSSAJ: Volume 72: Number 3 May June 2008

2 Table 1. Characteristics of the long-term fertility experiments (LTFEs) and the long-term continuous cropping experiment (LTCCE) at the Bicol Integrated Agricultural Research Center (BIARC), IRRI, and Philippine Rice Research Institute (PhilRice) in the Philippines. Parameter LTFEs BIARC IRRI PhilRice IRRI LTCCE Location Pili, Camarines Sur Los Baños, Laguna Muñoz, Nueva Ecija Los Baños, Laguna Rice crops per year Crop residue management Incorporated Incorporated Incorporated Removed Start of experiment First soil sampling Second soil sampling Soil classification Typic Pelludert Aquandic Epiaquoll Entic Pellustert Aquandic Epiaquoll Properties of the puddled top 20-cm layer Clay, g kg Sand, g kg Bulk density, Mg m ph (H 2 O), 1: Cation exchange capacity (CEC), cmol c kg Soil organic C (SOC), g kg Permanganate-oxidizable C, g kg Total N (N T ), g kg Stubble, which is about 60% of the total rice straw, was incorporated during land preparation. Cassman et al. (1996). Dobermann et al. (2000). Properties of soils sampled in 2000 (LTFEs) and 1998 (LTCCE), averaged across fertilizer treatments. Methods of analysis: texture by hydrometer, CEC by NH 4 OAc at ph 7, and SOC and N T by the combustion method using a CN analyzer. MATERIALS AND METHODS The Long-Term Experiments Measurements were taken from several treatments in four longterm experiments in the Philippines with contrasting fertilizer management, crop residue management, and cropping intensity (Table 1). The long-term fertility experiments (LTFEs), with two crops of rice per year, were conducted at three locations: Bicol Integrated Agricultural Research Center (BIARC, Camarines Sur, N, E), IRRI (Laguna, N, E), and the Philippine Rice Research Institute (PhilRice, Nueva Ecija, N, E). At each harvest, about 40% of the straw was removed with the grain, and the retained 60% of straw (also referred to as stubble i.e., the aboveground biomass remaining on the field after harvest) was incorporated during puddling (i.e., plowing and harrowing under flooded condition) and land preparation for the next rice crop. The long-term continuous cropping experiment (LTCCE) was conducted at IRRI with three rice crops per year, and all aboveground plant biomass was removed after each rice crop. At LTFE sites, treatments were a factorial combination of three rice cultivars and six to eight N P K fertilizer combinations arranged in a randomized complete block design with three (BIARC and PhilRice) or four (IRRI) replicates. In the LTCCE, the main plots were four rates of N divided into six subplots with different rice cultivars. All treatments in the LTCCE received fertilizer P and K. In all experiments, fertilizer P and K was basally incorporated into the soil and N was split-applied three or four times during the growing season. Zinc sulfate fertilizer was periodically applied to all plots in all experiments. Plot sizes were 18 to 21 m 2 at the LTFE sites and 69 m 2 in the LTCCE. All plots in the LTFEs and all main plots (fertilizer treatments) in the LTCCE were surrounded by permanent bunds to prevent transfer of soil and nutrients between plots. In all cases, rice was transplanted and grown on submerged soil with irrigation. Weeds and insects were controlled to avoid yield losses. The annual fertilizer N application averaged across the 15 yr in this study was 247 to 255 kg N ha 1 yr 1 in the LTFEs. In the LTCCE, average fertilizer N rates were 120, 240, and 359 kg N ha 1 yr 1 for the low-, medium-, and high-n treatments, respectively. In BIARC and IRRI (for both LTFEs and LTCCE), fertilizer P was 25 kg P ha 1 crop 1 and fertilizer K was 40 kg K ha 1 crop 1. The PhilRice LTFE received higher rates of P (30 kg P ha 1 crop 1 ) and K (100 kg K ha 1 crop 1 ). Soil at BIARC and PhilRice was classified as a Vertisol (Cassman et al., 1996) and the soil at IRRI (LTFE and LTCCE) was a Mollisol (Cassman et al., 1996; Dobermann et al., 2000) (Table 1). The soil texture was silty clay at PhilRice and clay at the other sites. Clay mineral analysis by x-ray diffraction revealed that the IRRI soil contained allophanes and amorphous minerals, while BIARC and PhilRice soils had a predominance of montmorillonite. The PhilRice soil also contained vermiculite and halloysite (Cassman et al., 1995; Dobermann et al., 1996). Soil Sampling Soil was collected with a core sampler at the start and end of a 15-yr interval (Table 1) from the 0- to 20-cm layer of puddled soil near the time of rice transplanting in the dry season. In the LTCCE, soil was also collected in 1991, midway in the 15-yr interval. For the LTFE sites, soil was obtained from plots for each of the three rice cultivars with the following four fertilizer treatments: no added N P K fertilizer, added N, added N P, and added N P K. For the LTCCE, soil was obtained from subplots for rice cultivars with four N rate treatments. All the soil collected from a plot was mixed thoroughly, subsampled, and air dried immediately after collection. Air-dried soil was crushed to pass a 2-mm sieve and stored in sealed plastic bottles at room temperature not exceeding 30 C. Soil bulk density was determined for the 0- to 20-cm layer at the time of the last sampling using a core ring (100-cm 3 volume, 5.1-cm height) and a bulk density sampler (Eijkelkamp Agrisearch Equipment, Giesbeek, the Netherlands). The four long-term experiments started between 1963 and 1968 (Table 1), but the samples collected in 1983 from the LTCCE and 1985 from the three LTFEs were the earliest available archived soil samples from the experiments. Soil samples collected after 8 and 15 yr from the LTCCE and after 15 yr from the LTFEs were processed and stored in SSSAJ: Volume 72: Number 3 May June

3 the same fashion as those from the initial sampling. Soil samples from all sampling times and all four experiments were concurrently retrieved from storage and prepared for analysis. Equal quantities of archived airdried soil from plots for all rice cultivars of the same sampling time and fertilizer treatment within a replicate were combined to form composite soil samples for each fertilizer treatment and replicate at each sampling time. All soil samples from all sampling dates and the four experiments were analyzed at the same time for a given analysis. In addition, soil profile samples were collected at 20-cm intervals to a depth of 80 cm during the fallow period after wet-season rice in Sampling was done with a 3.5-cm-diameter Edelman auger, except for the 0- to 20-cm layer in the IRRI LTFE that was sampled with cores (5.5-cm diameter) because the soil was puddled. For each soil layer, four individual soil samples were collected from each plot. For LTFE sites, soil samples were obtained from plots for one rice cultivar (IR72) with the following four fertilizer treatments: no added N P K fertilizer, added N K, added N P, and added N P K. For the LTCCE, soils were obtained from plots for one rice cultivar (IR72) with four N rate treatments. All collected soils from a plot were combined, homogenized, subsampled, and then air dried. Airdried soil was crushed to pass a 2-mm sieve and stored in sealed plastic bottles at room temperature (not exceeding 30 C) until analysis. Soil bulk density was determined at the time of soil sampling using a core ring (100-cm 3 volume, 5.1-cm height) positioned with a bulk density sampler (Eijkelkamp Agrisearch Equipment) in the middle portion of each 20-cm soil layer. Twelve measurements equally distributed among field replicates were obtained for each soil layer in each experiment. Soil and Plant Sampling and Analyses Permanganate-oxidizable C (POC) was determined by treating a 1-g soil sample (crushed to pass a 2-mm sieve) with 20 ml of mol L 1 KMnO 4, shaking for 15 min using a horizontal shaker, and covering the samples with aluminum foil or black plastic bags to exclude light during the oxidation procedure (Blair et al., 1995; Islam et al., 2003; Tirol-Padre and Ladha, 2004). The POC content of the soil was estimated from the amount of oxidized C, determined from absorbance at 565 nm. The POC provides an estimate of the active soil C that is more sensitive to management than total organic C (Shrestha et al., 2002; Islam et al., 2003). For the determination of ANM, 50 ml of deionized water was added to 10 g of air-dried soil, mixed, and preincubated at room temperature for 7 d in a sealed vessel with negligible air space. One set of samples was then extracted for determination of initial inorganic N, and a duplicate set of samples was incubated in a sealed vessel with negligible air space at 40 C for 7 d before determination of final inorganic N. Soil inorganic N was determined by extraction with 2 mol L 1 KCl (Keeney and Nelson, 1982) and subsequent colorimetric analysis for NH 4 N using the salicylate method (Kempers and Zweers, 1986). The ANM was calculated as the increase in NH 4 N during the 7-d incubation at 40 C. A subsample of each soil was ground to pass a 0.18-mm sieve. Total SOC and N T were determined on these subsamples by combustion with a CN analyzer. For each crop in the 15-yr period in each experiment, grain yield was determined from at least a 5-m 2 harvest area in each plot at harvestable maturity and reported at a standard water content of 140 g water kg 1 fresh weight. Samples of all aboveground plant biomass were also obtained from a six- or 12-hill sample for each plot and for each crop at physiological maturity. Grain and straw in these samples were separated and oven dried to constant weight at 70 C. Straw yield for each plot was estimated from the grain/straw ratio in the six- or 12-hill sample and the oven-dry grain yield in the 5-m 2 harvest area. Total dry matter in this study is the sum of grain yield (expressed at a water content of 30 g water kg 1 dry matter) and oven-dry straw yield. The concentration of total N in the grain and straw was determined by micro-kjeldahl digestion and subsequent titrimetric or colorimetric determination of NH 4 in most seasons of the 15-yr period in each experiment. Accumulations of N in grain (grain N) and straw (straw N) were calculated from the product of N concentration and dry weight. For seasons when N was not measured in grain or straw, grain N was estimated using measured relationships with grain yield for specific fertilizer treatments and experiments and straw N was estimated using measured relationships with straw yield for specific fertilizer treatments and experiments. Determination of Carbon and Nitrogen Balances We obtained C and N balances for the 15-yr period by using total C and N in the grain and straw for each cropping season, estimates of the fraction of the straw removed from the plots, recorded amounts of fertilizer N applied in each season, and measured SOC and N T in the top 20-cm soil layer at the start and end of the 15-yr period. The SOC and N T expressed in grams per kilogram were converted into kilograms per hectare by using the bulk density measured during the second sampling. The C returned to the soil with the aboveground rice residue was estimated using a plant C concentration of 410 g C kg 1, which was determined by CN analyzer for rice straw at crop maturity. This value is consistent with the value of 400 g C kg 1 reported by Ponnamperuma (1984). The estimated net C input represented the sum of C required from aquatic biomass, aboveground rice biomass, rice roots, and root exudates to maintain the measured SOC. The net C input was calculated as the sum of the estimated SOC mineralization and the measured change in soil C during the 15-yr period: Net C input= SOC mineralization+ change in soil C where net C input and change in soil C are expressed in kilograms of C per hectare per year, and SOC mineralization is the product of total SOC in the top 20-cm layer (kg C ha 1 ) and the fraction of this SOC mineralized annually. Based on earlier estimates by Bronson et al. (1997) of the annual C balance in the IRRI LTCCE, an estimated 4.3% of SOC was mineralized annually for treatments without fertilizer N and an estimated 4.9% of SOC was mineralized annually for treatments with fertilizer N. While the LTCCE had three rice crops per year with removal of all aboveground rice biomass, the LTFEs had two rice crops per year with incorporation of the rice straw (Table 1). Soil C mineralization could therefore differ between the LTFEs and the LTCCE because the addition of crop residues and frequency of soil tillage and cropping can influence C mineralization (Dao, 1998; Raiesi, 2006). In the absence of soil C mineralization data for the LTFEs, we estimated and compared net C input using a range for SOC mineralization (i.e., 3.5, 4.5, and 5.5% of total SOC in the top 20-cm layer per year). By comparison, SOM mineralization rates in the plow layer of double-cropped farmlands in northern China were within 2 to 5% (Wang et al., 1988), and microbial biomass in tropical rice soils accounts for 2 to 4% of total C (Reichardt et al., 2000). An N balance for the rice soil system to 20-cm soil depth was estimated from the net difference between the outputs and inputs of N in the system as reported by Ladha et al. (2000). The N outputs were the estimated export of N in grain and straw (i.e., crop removal) 800 SSSAJ: Volume 72: Number 3 May June 2008

4 Table 2. Changes in total soil organic C (SOC) and permanganate-oxidizable C (POC) in the top 20-cm soil layer during 15 yr of intensive, irrigated lowland rice cultivation with different fertilizer combinations for double rice cropping and crop residue incorporation averaged across three sites. Fertilizer SOC POC treatment Change Difference P > F Change Difference P > F g C kg g C kg 1 No N P K 18.3 b 20.0 b 1.7 a b 1.43 c 0.21 ab <0.001 N 19.7 a 21.4 a 1.7 a < a 1.70 a 0.33 a <0.001 N P 20.2 a 21.7 a 1.5 a a 1.60 ab 0.13 b N P K 20.5 a 21.3 a 0.7 a a 1.59 b 0.13 b Refers to the difference between the two sampling times. Within columns, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). and the change in soil N (i.e., increase or decrease in N T at the end of the 15-yr period) in the top 20-cm soil layer. Crop N removal was the sum of the N accumulation in grain and N accumulation in removed straw (40% in LTFEs and 100% in LTCCE). Nitrogen inputs included N from applied fertilizers and N from other sources such as irrigation water, rainfall, pesticides, and atmospheric deposition. The input of N from all sources other than fertilizer and biological N 2 fixation (BNF) was assumed to be 15 kg N ha 1 crop 1 (Ladha et al., 2000). Nitrogen losses by NH 3 volatilization, denitrification, leaching, and runoff were not included as N outputs in the N balance determination. In the absence of N fertilization, the N balance was assumed to represent N input by BNF. Data were analyzed using the Mixed procedure of SAS version (SAS Institute, 2003) to determine fertilizer treatment effects. A normality test was conducted for all data, and non-normal data were log-transformed before performing an ANOVA or mean comparison. For the LTFEs, a combined ANOVA was performed on data from the three sites, with site and fertilizer treatment as fixed effects and replicate as a random effect. The fertilizer treatment mean for the three sites was reported when there was no site fertilizer treatment interaction; otherwise, the fertilizer treatment mean for each site was presented. We conducted an ANOVA for each fertilizer treatment with sampling time (year) as a fixed effect and replicate as a random effect to test the significance of soil changes during 15 yr. Probability levels (P > F) are presented for assessing the difference in soil values between sampling dates at the start and end of the 15-yr interval. The LSMEANS test using the Tukey Kramer method was used to compare treatment means. Unless otherwise indicated, all mention of statistical significance refers to P < RESULTS AND DISCUSSION Properties of the Topsoil Table 1 shows soil properties determined from samples taken from the 0- to 20-cm layer near the time of rice transplanting in 2000 (LTFEs) and 1998 (LTCCE). The range in soil ph (H 2 O, 1:1) was 5.9 to 6.7, with the IRRI soils (LTFE and LTCCE) falling in the lower range and those in BIARC and PhilRice LTFEs in the upper range. The cation exchange capacity was higher in the IRRI soils ( 34 cmol c kg 1 ) than the PhilRice and BIARC soils ( 30 cmol c kg 1 ). The SOC and N T were lower at PhilRice than the other sites. The POC was highest at IRRI and lowest at PhilRice. Soil bulk density was not affected by fertilizer treatment, and means for the fertilizer treatments for puddled soil near the time of transplanting are reported in Table 1. Changes in Carbon and Nitrogen during Fifteen Years The application of fertilizer in the LTFEs had a significant positive effect on SOC, POC, and N T at both the start and end of the 15-yr period (Tables 2, 3, 4, and 5). In the LTFEs, levels were consistently higher with N P K than with no N P K (Tables 2 and 4). In the LTCCE, levels were also consistently higher with high N than no N (Tables 3 and 5). Fertilization resulted in greater plant growth leading to more inputs of C from straw and roots, which presumably contributed to the higher SOC and N T with fertilization. The SOC, POC, and N T in the top 20-cm soil layer increased or remained the same after 15 yr of intensive cultivation for both double rice cropping with crop residue incorporation (LTFEs) and triple rice cropping with all crop residue removed (LTCCE) (Tables 2, 3, 4, and 5). The SOC, POC, and N T were never significantly lower in the last than the first soil sampling. In the LTCCE, the concentrations of SOC, POC, and N T in 1991 were consistently intermediate between those in the 1983 and 1998 samplings (Tables 3 and 5). The change in SOC and N T during 15 yr was not affected by fertilizer N P K combinations in the LTFEs and the different levels of N in the LTCCE (Tables 2, 3, 4, and 5). The change in POC in the LTFEs, however, was greater in the treat- Table 3. Changes in total soil organic C (SOC) and permanganate-oxidizable C (POC) in the top 20-cm soil layer during 15 yr of intensive, irrigated lowland rice cultivation with different fertilizer combinations for triple rice cropping with all crop residue removed. SOC POC Fertilizer Change Change treatment Difference P > F Difference P > F g C kg 1 - g C kg 1 - No N 22.0 b 22.7 b 23.5 b 1.5 a c 1.84 b 1.99 b 0.38 a Low N 22.2 b 23.1 b 24.5 ab 2.3 a bc 1.96 ab 2.04 b 0.31 a Medium N 23.5 ab 24.3 ab 25.5 ab 2.0 a ab 2.03 ab 2.21 ab 0.34 a High N 24.9 a 26.2 a 26.6 a 1.7 a a 2.23 a 2.36 a 0.33 a All treatments received a basal application of P and K every cropping season. Refers to the difference between the sampling in 1998 and Within columns, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). SSSAJ: Volume 72: Number 3 May June

5 Table 4. Changes in total N (N T ) and anaerobic N mineralization (ANM) in the top 20-cm soil layer during 15 yr of intensive, irrigated lowland rice cultivation with different fertilizer combinations for double rice cropping and crop residue incorporation averaged across three sites. Fertilizer N T ANM treatment Change Difference P > F Change Difference P > F g N kg 1 - mg N kg 1 d 1 No N P K 1.49 b 1.67 b 0.18 a b a N 1.63 a 1.84 a 0.21 a < a a N P 1.69 a 1.89 a 0.21 a < a a N P K 1.72 a 1.84 a 0.13 a a a Refers to the difference between the two sampling times. Within columns, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). There was a significant site treatment interaction for ANM of soils sampled in 2000 at the three long-term fertility experiment sites; ANM was significantly lower (0.05) for no N P K fertilizer than other treatments at IRRI and PhilRice but not at BIARC. ment receiving fertilizer N alone (Table 2). This is consistent with short-term increases in POC observed by Shrestha et al. (2002) when crop residues were applied with N rather than applied alone. In the LTCCE, fertilizer N rate had no effect on the change in POC during 15 yr (Table 3). Shrestha et al. (2002) similarly observed no effect of fertilizer N on POC in the absence of crop residues. The effect of fertilizer N with crop residue incorporation on increasing POC could be associated with narrowing the C/N ratio of the crop residue, which hastens its decomposition, but the advantage of N alone compared with N P and N P K (Table 2) is unclear. In 1985, ANM was significantly higher with fertilization than with no N P K across the three LTFE sites (Table 4); a similar trend was present in 2000 (Table 4) except for the BIARC site, where there was no significant difference among fertilizer treatments (data not shown). A comparison of the two sampling times showed that ANM declined during 15 yr without N P K and with N alone but not in the N P and N P K treatments (Table 4). The change in ANM during 15 yr was, however, not affected by N P K fertilizer treatment. In the LTCCE, where all crop residue was removed, ANM was not affected by N fertilization in 1983 and 1991 (Table 5), but in 1998 ANM was higher with fertilizer N than without fertilizer N. This is consistent with observations in China of higher N mineralization with N fertilization (Yan et al., 2006). The ANM was statistically similar between the 1983 and 1998 samplings, indicating no significant decline in ANM during 15 yr (Table 5). The SOC, POC, and N T in these rice soils, regardless of fertilizer treatment, were maintained or even improved regardless of whether aboveground rice residue was incorporated (LTFEs) or completely removed (LTCCE). The results indicate that even with complete removal of aboveground rice residues, root residues and organic inputs from aquatic biomass mainly algae (Roger, 1996) in the soil floodwater ecosystem can sustain C and N of soil not exposed to prolonged drying. Carbon and Nitrogen Balances Carbon Assuming an annual SOC mineralization rate of 4.3% without fertilizer N (N 0 ) and 4.9% with fertilizer N (N f ) (Bronson et al., 1997), the net C input which represents the estimated net input of C from aquatic biomass, stubble, roots, and root exudates ranged from 1181 to 1597 kg C ha 1 yr 1 without fertilizer N and from 1414 to 1899 kg C ha 1 yr 1 with fertilizer N (Table 6). The estimated net C input to the top 20-cm soil layer was about 100 to 400 kg C ha 1 yr 1 higher with N fertilization than without N fertilization for the experiments. The net annual C input at IRRI was higher for the LTCCE with three crops per year than for the LTFEs with two crops per year. When estimated on a crop basis, the estimated net C input for the two experiments at IRRI was comparable and in the range of 500 to 600 kg C ha 1 crop 1 without fertilizer N and 600 to 700 kg C ha 1 crop 1 with fertilizer N. The estimated net C input increased as the assumed SOC mineralization rate increased (Table 6). When the same mineralization rate of total soil C was assumed across fertilizer treatments, the estimated net C inputs at a given site were comparable for treatments with and without fertilizer N because the change in soil C during 15 yr was not strongly influenced by fertilizer N (Table 6). Regardless of assumed mineralization rate for SOC, considerable net C input was required to maintain the C balance in the IRRI LTCCE in which all aboveground rice biomass was removed for every crop. These results suggest that root residues and exudates and aquatic biomass in the soil Table 5. Changes in total N (N T ) and anaerobic N mineralization (ANM) in the top 20-cm soil layer during 15 yr of intensive, irrigated lowland rice cultivation with different fertilizer combinations for triple rice cropping with all crop residue removed. N Fertilizer T ANM treatment Change Change Difference P > F Difference P > F g N kg mg N kg 1 d No N 1.92 b 1.99 b 2.01 c 0.09 a a 19.4 a 18.8 b 2.7 b Low N 1.92 b 2.00 b 2.07 bc 0.15 a a 19.1 a 22.9 a 2.5 a Medium N 2.03 ab 2.15 ab 2.17 ab 0.14 a a 21.0 a 22.9 a 0.8 ab High N 2.15 a 2.25 a 2.26 a 0.12 a a 20.7 a 24.9 a 2.9 a All treatments received a basal application of P and K every cropping season. Refers to the difference between the sampling in 1998 and Within columns, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). 802 SSSAJ: Volume 72: Number 3 May June 2008

6 Table 6. Annual C balances to 20-cm soil depth during 15 yr of intensive, irrigated lowland rice cultivation in double rice cropping with crop residue incorporation (LTFEs) and triple rice cropping with all crop residue removed (LTCCE) at Bicol Integrated Agricultural Research Center (BIARC), IRRI, and Philippine Rice Research Institute (PhilRice) in the Philippines. Site and experiment Crops per year Mean SOC Change in soil C Aboveground plant C returned Estimated net C input for different rates of annual SOC mineralization 4.3% (N 0 ), 4.9% (N f ) 3.5% 4.5% 5.5% Mg C ha kg C ha 1 yr Without fertilizer N (N 0 )# BIARC LTFE a 170 a 1567 a 1597 a 1332 a 1664 a 1995 a IRRI LTFE b 0 a 1454 a 1181 a 961 a 1236 a 1510 a PhilRice LTFE b 439 a 1286 b 1591 a 1377 a 1645 a 1913 a IRRI LTCCE a 136 a a 1235 a 1549 a 1863 a With fertilizer N (N f ) BIARC LTFE a 104 a 2593 c 1866 a 1362 a 1722 a 2081 a IRRI LTFE b 63 a 2750 b 1414 b 992 b 1294 b 1595 b PhilRice LTFE b 259 a 2878 a 1706 ab 1293 ab 1588 ab 1883 ab IRRI LTCCE a 156 a a 1401 a 1757 a 2113 a Mean soil organic C during the 15-yr period based on a soil bulk density of 0.71 Mg m 3 at BIARC, 0.65 Mg m 3 at IRRI LTFE, 1.04 Mg m 3 at PhilRice, and 0.69 Mg m 3 at IRRI LTCCE. Bulk density was not affected by fertilizer treatment. Based on a C concentration of 410 g kg 1 for stubble incorporated at the LTFE sites (i.e, 60% of straw yield at harvest). All aboveground residues were removed at IRRI LTCCE. Net C input = SOC mineralization + change in soil C, representing the net C input from aquatic biomass, stubble, roots, and root exudates required to maintained measured soil C. SOC mineralization is based on an assumed annual turnover of 4.3% of total soil C without fertilizer N (N 0 ) and 4.9% of total soil C with fertilizer N (N f ) (Bronson et al., 1997). # Refers to no N P K treatment in the LTFEs and no N treatment in the LTCCE. Within columns and fertilizer treatments, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). Refers to N P K treatment in the LTFEs and high-n treatment in the LTCCE. floodwater ecosystem can be important sources of soil C in irrigated rice systems with submerged soils. Bronson et al. (1997) estimated maximum root biomass at rice maturity to be 19% of the total aboveground biomass without fertilizer N and 14% of the total aboveground biomass with fertilizer N. Roger (1996) reported daily primary production of aquatic biomass ranging from 2 to 12 kg C ha 1 d 1 at IRRI, which suggests that aquatic biomass mainly algae can represent an important C input for submerged soils with continuous cultivation of rice. Nitrogen Total soil N remained unchanged or slightly increased during 15 yr of intensive rice cultivation without fertilizer N (Tables 4, 5, and 7) and with N P K fertilization (Tables 4, 5, and 8). Nonsymbiotic BNF as estimated from N balances in the absence of fertilizer N ranged from 37 to 88 kg N ha 1 yr 1 or 19 to 44 kg N ha 1 crop 1 (Table 7). These estimates highlight the important role of BNF in maintaining N in submerged rice soils (Roger and Ladha, 1992; Ladha et al., 2000). The contribution of BNF would be greater if N losses occurred because the estimates assume no gaseous, leaching, or runoff losses of N. Roger and Ladha (1992) reported N balances of 19 to 98 kg N ha 1 crop 1 in longterm experiments with no fertilizer N application. Likewise, Koyama and App (1979) estimated an N input from BNF of 15 to 50 kg N ha 1 crop 1. The N balances indicate large net losses of N when moderate to high rates of fertilizer N were applied (Table 8), suggesting that N losses from fertilizers in such cases far exceeded N inputs by BNF. A positive N balance, indicating greater input by BNF than loss of fertilizer N, only occurred at a relatively low rate of N fertilization at the IRRI LTCCE (Table 8). Relatively large losses of applied fertilizer N, as indicated by the N balances, have been well documented for submerged rice soils (De Datta et al., 1988; Buresh and De Datta, 1990; Buresh et al., 2008). Table 7. Annual N balances to 20-cm soil depth during 15 yr of intensive, irrigated lowland rice cultivation without N fertilizer in double rice cropping with crop residue incorporation (LTFEs) and triple rice cropping with all crop residue removed (LTCCE) at Bicol Integrated Agricultural Research Center (BIARC), IRRI, and Philippine Rice Research Institute (PhilRice) in the Philippines. Site and experiment Crops per year Change in soil N (A) Crop removal (B) Other inputs (C) Annual N balance [(A + B) C] Recycled with straw kg N ha 1 yr BIARC LTFE 2 19 ab 80 b ab 22 a IRRI LTFE 2 5 b 72 bc b 21 a PhilRice LTFE 2 54 a 64 c a 18 b IRRI LTCCE 3 9 b 117 a a 0 Assumes an input of 15 kg N ha 1 crop 1 from other sources such as irrigation water, rainfall, pesticides, and atmospheric deposition (Ladha et al., 2000). Estimated N input from biological N 2 fixation. This N balance assumes no N losses by NH 3 volatilization, denitrification, leaching, or runoff. 60% of the straw returned at the LTFE sites; all aboveground residues removed at IRRI LTCCE. Within columns, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). SSSAJ: Volume 72: Number 3 May June

7 Table 8. Annual N balances to 20-cm soil depth during 15 yr of intensive, irrigated lowland rice cultivation with N fertilizer in double rice cropping with crop residue incorporation (LTFEs) and triple rice cropping with all crop residue removed (LTCCE) at Bicol Integrated Agricultural Research Center (BIARC), IRRI, and Philippine Rice Research Institute (PhilRice) in the Philippines. Site and treatment Crops per year Change in soil N (A) Crop removal (B) Fertilizer input (D) Other inputs (C) Annual N balance [(A + B) (C + D)] Recycled with straw kg N ha 1 yr 1 LTFE N P K BIARC 2 16 a 160 c a 50 c IRRI 2 4 a 185 a a 68 a PhilRice 2 35 a 173 b a 59 b IRRI LTCCE Low N 3 13 a 167 c a 0 Medium N 3 13 a 224 b b 0 High N 3 11 a 260 a c 0 Assumes an input of 15 kg N ha 1 crop 1 from other sources such as irrigation water, rainfall, pesticides, and atmospheric deposition (Ladha et al., 2000). Net difference between inputs from biological N 2 fixation and outputs through N losses. 60% of the straw returned at the LTFE sites; all aboveground residues removed at IRRI LTCCE. Within columns and experiments, means followed by the same letter are not significantly different according to Tukey-Kramer (0.05). Carbon and Nitrogen in the Soil Profile In the LTFEs, SOC and N T in the topsoil (0 20-cm layer) were higher following long-term cropping with N K, N P, or N P K fertilization than with no fertilization (Tables 9 and 10). Significant treatment differences in topsoil POC were observed at BIARC and PhilRice but not at IRRI (Table 11). Topsoil ANM tended to be higher in fertilized than in unfertilized treatments but a significant difference was observed only between no N P K and N P at BIARC and PhilRice (Table 11). In the LTCCE, N fertilization significantly increased POC in the topsoil (Table 9), but it did not significantly increase SOC, N T, or ANM even though values tended to be higher with N fertilization (Tables 9 and 10). This is consistent with previous findings that POC is more sensitive than SOC to short-term effects of management practices (Shrestha et al., 2002; Islam et al., 2003). Below the plow layer (i.e., cm depth) of the LTFEs, a significant treatment effect was observed only for POC at 20 to 40 cm, where POC was higher with N P than N P K (Table 9). Subsoil SOC, N T, and ANM were not affected by fertilizer treatments (Tables 9 and 10). Likewise, fertilizer N application in the LTCCE did not increase SOC, N T, POC, or ANM in the subsoil (Tables 9 and 10). At PhilRice, negative values of ANM were observed in the subsoil (data not shown), reflecting lower NH 4 N after than before the 7-d anaerobic incubation at 40 C. Extractable NH 4 N before the 7-d anaerobic incubation i.e., after the 7-d preincubation at room temperature could have been derived from killed soil microorganisms and decomposed organic substances resulting from prior soil air drying (Sparling and Ross, 1988). The decrease in NH 4 N after the 7-d anaerobic incubation is indicative of NH 4 fixation by 2:1 silicate minerals, especially vermiculite (Brady and Weil, 2002), which is present in the PhilRice soil (Cassman et al., 1995). The SOC, POC, N T, and ANM decreased with increasing soil depth in all experiments (Tables 9 and 10). Similarly, POC, expressed as a percentage of SOC, decreased with soil depth regardless of fertilizer treatment and experiment (Fig. 1). This is consistent with other observations of greater influences of crop and residue management on POC in topsoil than subsoil (Shrestha et al., 2006). In the LTFEs, POC as a percentage of SOC was highest for N P at 0 to 20 and 20 to 40 cm (Fig. 1). In the LTCCE, the long-term application of high fertilizer N with removal of all aboveground crop residues increased POC as a percentage of SOC in the topsoil, suggesting that roots contribute to POC. Table 9. Soil organic C (SOC) and permanganate-oxidizable C (POC) in the soil profile after >30 yr of continuous rice cultivation with different fertilizer combinations in double rice cropping with crop residue incorporation (LTFEs) and triple rice cropping with all crop residue removed (LTCCE). Treatment SOC POC 0 20 cm cm cm cm 0 80 cm 0 20 cm cm cm cm 0 80 cm Mg C ha 1 Mg C ha 1 LTFEs (across three sites) No N P K 26.7 b 21.5 a 12.7 a 10.7 a 71.6 a 1.47 ab 0.76 a 0.65 a 4.92 ab N K 29.3 a 20.2 a 13.4 a 11.1 a 73.9 a 1.42 ab 0.85 a 0.72 a 5.19 a N P 31.3 a 23.3 a 13.8 a 10.7 a 79.1 a 1.67 a 0.89 a 0.67 a 5.71 a N P K 30.0 a 19.9 a 12.2 a 10.1 a 72.1 a 1.29 b 0.72 a 0.61 a 4.89 b LTCCE No N 36.4 a 27.4 a 19.9 a 12.9 a 96.7 a 2.97 b 2.14 a 1.25 a 0.79 a 7.17 a Low N 39.4 a 30.5 a 21.1 a 14.5 a a 3.32 ab 2.36 a 1.35 a 0.89 a 7.92 a Medium N 38.7 a 30.1 a 22.5 a 17.3 a a 3.29 ab 2.38 a 1.46 a 1.03 a 8.15 a High N 41.9 a 30.7 a 21.2 a 13.0 a a 3.70 a 2.44 a 1.37 a 0.72 a 8.23 a Within columns and experiments, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). All treatments received a basal application of P and K every cropping season. 804 SSSAJ: Volume 72: Number 3 May June 2008

8 Table 10. Total nitrogen (N T ) and anaerobic N mineralization (ANM) in the soil profile after >30 yr of continuous rice cultivation with different fertilizer combinations in double rice cropping with crop residue incorporation (LTFEs) and triple rice cropping with all crop residue removed (LTCCE). Treatment N T ANM 0 20 cm cm cm cm 0 80 cm 0 20 cm cm cm cm Mg N ha 1 kg N ha 1 d 1 LTFEs (across three sites) No N P K 2.09 c 1.61 a 0.95 a 0.83 a 5.48 b 7.30 a 1.74 a 1.08 a N K 2.35 b 1.55 a 1.02 a 0.86 a 5.77 ab 7.10 a 1.98 a 1.41 a N P 2.59 a 1.82 a 1.05 a 0.84 a 6.30 a 7.95 a 2.26 a 1.28 a N P K 2.46 ab 1.50 a 0.96 a 0.80 a 5.72 ab 5.58 a 1.18 a 0.88 a LTCCE No N 3.10 a 2.08 a 1.33 a 0.90 a 7.41 a 20.1 a 8.3 a 2.8 a 0.02 a Low N 3.32 a 2.36 a 1.43 a 0.99 a 8.10 a 21.4 a 10.2 a 3.2 a 1.14 a Medium N 3.25 a 2.30 a 1.52 a 1.10 a 8.16 a 21.4 a 8.6 a 3.0 a 0.80 a High N 3.46 a 2.26 a 1.41 a 0.87 a 8.01 a 20.9 a 9.1 a 2.8 a 0.21 a Values for ANM show the average for two LTFE sites only, BIARC and IRRI; PhilRice data were excluded because of negative ANM values in the subsoil. Within columns and experiments, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). All treatments received a basal application of P and K every cropping season. The effect of N P K fertilization on SOC, POC, N T, and ANM in the topsoil was examined from the difference between the full N P K treatment and a control (i.e., full N P K minus no N P K for the LTFEs and high N minus no N for the LTCCE) (Fig. 2). The effect of N P K fertilization was especially pronounced in the IRRI and PhilRice LTFEs. Full N P K fertilization increased crop growth and the amount of recycled above- and belowground crop residues, which in turn influenced soil C and N. The effect of N P K fertilization was, however, confined to the topsoil; SOC and N T in the subsoil (20 80 cm) was not affected by fertilization. Long-term N fertilization in dryland cropping systems has similarly been shown to increase the quantity of crop residues but have no effect on the SOC content of the subsoil (Halvorson et al., 2002). Continuous intensive rice cropping can conceivably increase SOC in the subsoil because the application of rice residues can increase dissolved C in percolating water in rice fields (Katoh et al., 2005). Long-term changes in subsoil C and N cannot be definitively assessed in our study with only a one-time determination of SOC and N T in the soil profile. The absence of a fertilization effect on subsoil SOC and N T after 32 to 37 yr of continuous rice cropping nonetheless suggests that the effect of rice cropping on the accumulation of SOC and N T below the topsoil (i.e., plow layer) was small or negligible. General Discussion The SOM was maintained or even slightly increased during long-term cropping with two or three rice crops per year with near-continuous soil submergence, irrespective of fertilizer and Table 11. Permanganate-oxidizable C (POC) and anaerobic N mineralization (ANM) in the topsoil (0 20 cm) after >30 yr of continuous rice cultivation with different fertilizer combinations in double rice cropping with crop residue incorporation (LTFEs) at Bicol Integrated Agricultural Research Center (BIARC), IRRI, and Philippine Rice Research Institute (PhilRice) in the Philippines. Treatment POC ANM BIARC IRRI PhilRice BIARC IRRI PhilRice Mg C ha 1 - kg N ha 1 d 1 No N P K 1.99 b 2.16 a 1.96 b 13.2 b 11.5 a 4.4 b N K 2.00 ab 2.43 a 2.18 b 14.5 ab 17.7 a 8.7 ab N P 2.42 a 2.41 a 2.63 a 21.4 a 14.3 a 13.2 a N P K 2.09 ab 2.37 a 2.32 ab 15.2 ab 16.6 a 10.9 ab Within columns, means followed by the same letter are not significantly different according to Tukey Kramer (0.05). Fig. 1. Permanganate-oxidizable C (POC) expressed as a percentage of total soil organic C (SOC) at different soil depths and fertilizer treatments after more than 30 yr of continuous rice cultivation in double rice cropping with crop residue incorporation (LTFE) and triple rice cropping with all crop residue removed (LTCCE). Within a soil layer, bars with the same letter are not significantly different according to Tukey Kramer (0.05). SSSAJ: Volume 72: Number 3 May June

9 Fig. 2. Effect of long-term NPK fertilization on (a) soil organic C (SOC), (b) permanganate-oxidizable C (POC), (c) total N (N T ), and (d) anaerobic N mineralization (ANM) at the topsoil (0 20 cm) with double-cropping and crop residue incorporation (LTFE), and triple-cropping with all crop residue removed (LTCCE) at the Bicol Integrated Agricultural Research Center (BIARC), IRRI, and Philippine Rice Research Institute (PhilRice) in the Philippines. Values shown are differences between N P K fertilization treatment and the control (no N P K for LTFE and no N for LTCCE). * Significant at the 0.05 probability level; ** significant at the 0.01 probability level; NS = not significant. crop residue management. These findings are consistent with other reports of SOM maintenance and increase with continuous rice cropping on submerged soils (Sahrawat, 2004; Li et al., 2005). In addition, our study shows that the return of aboveground crop residue is not essential to maintain or even slightly increase SOM in submerged rice soils (Tables 2, 3, 4, and 5). Factors contributing to the maintenance of SOC and N T even in the absence of fertilizer N and crop residues include slower C mineralization in submerged than aerated soil (Acharya, 1935), substantial inputs of C from aquatic biomass in submerged rice fields (Roger, 1996; Bronson et al., 1997), and greater inputs of N via nonsymbiotic BNF in submerged than aerated soil (Roger and Ladha, 1992). Our C balances (Table 6) confirm net C inputs of which aquatic biomass was presumably a major source, and our N balances (Table 7) confirm the findings of others (Ladha et al., 2000) of substantial inputs of N via BNF. Topsoil SOC and N T were higher with full N P K fertilization than without fertilizer in the LTFEs and without fertilizer N in the LTCCE, presumably because of enhanced crop growth and concomitant inputs of C with full fertilization. The effect of fertilization on SOC and N T occurred before the first sampling (Tables 2, 3, 4, and 5), which was after 17 to 21 yr of continuous rice cultivation. During the subsequent 15 yr of continuous rice cropping, fertilizer management had no differential effect on topsoil SOC and N T. Our results suggest that beneficial effects of additional C inputs arising from increased crop growth with full N P K fertilization already occurred by 17 to 21 yr of continuous rice cropping, resulting in a steady state for the relative difference in SOM content among fertilizer regimes. These differences in SOM content among fertilizer management practices were then maintained through additional rice cropping, and subsequent changes in SOC and N T were comparable among fertilizer practices. Continuous or near-continuous soil submergence, resulting in depletion of O 2, is a critical factor contributing to the maintenance of SOM in intensive, continuous rice cropping. Prolonged soil submergence can also promote the buildup of less-decomposed substances, which become incorporated into young SOM fractions. Olk et al. (1996, 2000) observed, in these same long-term experiments with continuous rice cropping, a buildup of phenolic compounds originating from less humification. This led to a concern that these compounds could immobilize N abiotically, thereby reducing the rate of net N mineralization in submerged soils and adversely impacting the sustainability of intensive lowland rice production (Schmidt-Rohr et al., 2004). In our study, ANM was used as an integrative measure for the net effect of the numerous soil biological and chemical processes influencing the supply of plant-available N. In the absence of full N P K fertilization, ANM declined slightly during the 15 yr of continuous rice cropping (Table 4). With full N P K fertilization, however, ANM remained relatively high and stable during 15 yr of continuous rice cropping with near-continuous soil submergence (Tables 4 and 5). We cannot, from our study, directly assess abiotic immobilization of soil N in aromatic rings as proposed by Schmidt-Rohr et al. (2004), and we cannot ascertain the effect on ANM of the initial 17 to 21 yr of continuous rice cropping before the first soil sampling. We nonetheless found consistently no evidence across four experiments for a net decline in soil N-supplying capacity during 15 yr of continuous rice cropping with N P K fertilization. Our findings suggest that submerged soils can sustain a long-term N-supplying capacity even when cultivated intensively with two and three rice crops per year. ACKNOWLEDGMENTS We thank the following, who managed the long-term experiments during the period covered in this study: Mr. Josue Descalsota and Mr. Joven Alcantara for the LTFE and LTCCE, respectively, at IRRI ; Ms. Eliza Imperial for the BIARC LTFE, ; and Mr. Wilfredo Collado for the PhilRice LTFE. We also thank Ms. Evelyn 806 SSSAJ: Volume 72: Number 3 May June 2008