23-year manure and fertilizer application increases soil organic carbon sequestration of a rice–

ORIGINAL PAPER23-year manure and fertilizer application increases soil organic carbon sequestration of a rice–barley cropping systemYidong Wang1&Ning Hu2&Minggang Xu3&Zhongfang Li4&Yilai Lou2,5&Yi Chen6&Chunyan Wu6&Zhong-Liang Wang1Received:24November2014/Revised:11March2015/Accepted:17March2015#Springer-Verlag Berlin Heidelberg2015Abstract Fertilizer application can potentially change soil or-ganic C(SOC)sequestration of croplands.However,how fer-tilization affects SOC sequestration and soil C fractions has not been sufficiently investigated.Here,we studied this issue in a rice–barley system of eastern China after23-year repeated organic and chemical fertilizer amendments.The fertilization treatments were as follows:unfertilized control(CK),pig ma-nure(M),chemical fertilizer NPK(NPK),and pig manure combined with NPK(MNPK).Our results showed that M, NPK,and MNPK significantly increased25,13,and30% of SOC content as compared with pared with CK, the three fertilizations,especially manure addition,significant-ly increased the content of SOC fractions such as microbial biomass C,dissolved organic C,labile organic C,and particulate organic pared with the initial SOC stock at0–20-cm depth(41.2Mg C ha−1),M,NPK,and MNPK increased11,1,and16%of SOC storage,respectively, whereas CK decreased9%of SOC pool.The SOC seques-tration was positively correlated with the cumulative C input (root residue-C+manure-C)(SOC sequestration=0.21×C input−5.67,R2=0.88,P<0.001).This linear relationship suggested that(i)the soil had not reached a SOC saturation, (ii)the conversion rate from input-C to SOC was21%,and (iii)a C input threshold of1.17Mg C ha−1year−1was needed for maintaining the initial SOC level.Therefore,manure ap-plication is recommended for promoting SOC sequestration in the rice–barley cropping system of eastern China. Keywords Fertilization.Manure amendment.Soil carbon dynamic.Soil carbon sequestrationIntroductionSoil organic C(SOC)is of importance to maintain soil fertility and to sustain productivity of agro-ecosystems(Su et al.2006; Kundu et al.2007).In the context of climate change,SOC also plays an important role in global C cycling(Schimel1995). Therefore,it is significant to keep a satisfactory level of SOC in agro-ecosystems for ensuring food security and mitigating climate warming.In agro-ecosystems,rhizodeposition,residues of crop root and stubble,and organic fertilizer are main C input forms, while mineralization is the main C output.According to the status and stability,SOC has several sensitive fractions:mi-crobial biomass C(MBC),dissolved organic C(DOC),labile organic C(LOC)(with rapid turnover rates),and particulate organic C(POC).The dynamics of SOC and its fractions depends on various agricultural managementstrategies, Yidong Wang and Ning Hu contributed equally to this work.*Zhongfang Lilizhongfang08@*Yilai Loulouyilai@1Tianjin Key Laboratory of Water Resources and Environment,Tianjin Normal University,Tianjin300387,China2Institute of Environment and Sustainable Development inAgriculture,Chinese Academy of Agricultural Sciences,100081Beijing,China3Institute of Agricultural Resources and Regional Planning,ChineseAcademy of Agricultural Sciences,100081Beijing,China4Chemistry and Bioengineering College,Hezhou University,Hezhou542899,China5State Key Laboratory of Forest and Soil Ecology,Institute of AppliedEcology,Chinese Academy of Sciences,Shenyang110016,China6Institute of Environmental Resources and Soil Fertilizer,ZhejiangAcademy of Agricultural Sciences,Hangzhou310021,ChinaBiol Fertil SoilsDOI10.1007/s00374-015-1007-2especially fertilization(e.g.,Kong et al.2005;Lou et al. 2011a;Malhi et al.2011).However,contradictory effects of chemical fertilizer application on SOC trends have been ob-served.Both positive impacts(Verma and Sharma2007; Purakayastha et al.2008;Gong et al.2009;Brar et al.2013) and no influences(Russell et al.2005;Manna et al.2006; Rudrappa et al.2006;Lou et al.2011b)of chemical fertiliza-tion on SOC sequestration and its fractions have been report-ed.The inconsistent results of SOC sequestration may be at-tributed to differences in crop residue incorporations(Malhi et al.2011),fertilizer application rate,tillage regime,and ex-periment duration(Raun et al.1998;Su et al.2006).In com-parison to chemical fertilizer,organic fertilizer was more effi-cient in SOC sequestration due to the organic C input (Purakayastha et al.2008;Yu et al.2012;Zhang et al.2012, 2014).Even though fertilization-induced changes in SOC have been reported,the relative mechanisms of SOC sequestration to fertilization-derived C input(organic fertilizer+crop resi-dues)were poorly understood.A linear relationship between the SOC sequestration and C input has been shown(e.g., Kong et al.2005;Kundu et al.2007;Zhang et al.2010;Fan et al.2014).Nevertheless,some soils with high SOC levels show little or no increase in SOC pool with twofold to three-fold increases in C inputs(Campbell et al.1991;Gulde et al. 2008),and this has been generally explained by the SOC saturation level(Six et al.2002;Stewart et al.2009).The SOC unsaturated soils have linear relationships between the SOC sequestration and C input,and this is not the case of SOC saturated soils.Therefore,it is important to quantify the rela-tionship between the SOC sequestration and C input.The plain of lower reaches of the Yangtze River is one of the most important agricultural regions in China.And,the zones are mainly subjected to the paddy upland rotation and generally fertilized with organic and chemical fertilizers to ensure high grain yields(Kölbl et al.2014).However,it is still unclear how fertilization affects SOC sequestration in the zones of this region.Here,we studied this issue using a 23-year field experiment in a rice–barley rotation system with repeated organic and chemical fertilizer amendments.The objective of this study was to quantify the23-year effects of manure and chemical fertilizer application on SOC stock as well as its LOC fractions,to understand mechanism of SOC sequestration.Materials and methodsStudy siteThis study was conducted in a rice–barley field(30°26′04″N,120°25′01″E,elevation3–4m)at the National Monitoring Station for Soil Fertility and Fertilizer Efficiency in Hangzhou,Zhejiang Province,eastern China.The study site is flat and well drained.The region is characterized by a subtropical humid monsoon climate,with a mean air temper-ature of16–17°C and an annual rainfall of1500–1600mm. Annual evapotranspiration is1000–1100mm year−1.Mean annual frost-free period and sunshine duration hours are 240–250days and1900–2000h,respectively.The soil,clas-sified as an Inceptisol according to the soil taxonomy of US Department of Agriculture(Chen et al.2010a),has a loam texture with42%sand,38%silt,and20%clay(Chen et al.2010b).The formal long-term field experiment was launched in autumn1990.Prior to the experiment,the land had been in-tensively cultivated for at least30years with chemical fertil-izer amendment,and afterward,the field was homogenized by growing barley,early rice,and late rice in rotation for2years (from autumn1988to autumn1990)without fertilizer addi-tion.The main properties of the initial surface soil(0–20cm) in1990were as follows:bulk density,1.24g cm−3;porosity, 53.2%;TOC,16.6g kg−1;total N,1.67g kg−1;total P, 2.53g kg−1;available N,94.1mg kg−1;available P, 37.4mg kg−1;available K,67.5mg kg−1;CEC, 14.6cmol kg−1;and pH,6.4.Experimental designFour fertilizer application regimes,unfertilized control(CK), pig manure(M),chemical fertilizer NPK(NPK),and pig ma-nure combined with NPK(MNPK),were arranged in a completely random design with three replicates per each treat-ment.The fresh pig manure,with68.9%water content,was applied with an annual rate of22.5Mg ha−1year−1.The dry matter of pig manure had a C content of197.4g kg−1,N content of14.5g kg−1,P concentration of14.2g kg−1,and K concentration of13.1g kg−1.Thus,the annual application rates of C,N,P,and K of pig manure are1.4Mg C ha−1year−1, 101kg N ha−1year−1,228kg P2O5ha−1year−1,and110.6kg K2O ha−1year−1,respectively.N,P,and K fertilizations were applied as urea(46%N),calcium superphosphate (CaP2H4O8,16%P2O5),and potassium chloride(KCl, 60%K2O),at rates of315kg N ha−1year−1,157.5kg P2O5 ha−1year−1,and157.5kg K2O ha−1year−1,respectively.The area of each replicated plot was100m2.During the first10years(from autumn1990to autumn2000),a rotation of barley-early rice-late rice was selected for annual cropping. The percentage quotas of fertilizer application with respect to the annual rate were20%for barley,40%for early rice,and 40%for late rice.After10years,soils were cropped to bar-ley–rice rotation,and the percentages of applied fertilizers were32%for barley and68%for rice.In each single growing season,the annual rate of manure,P,and K and70%of the N fertilization rate were applied as base fertilizer;while the re-maining30%N was used as top dressing.The other fieldBiol Fertil Soilsmanagement practices were identical among different treat-ments.The barley was seeded in the late November and har-vested in the early May of the next year across the23years.In the three annual crop rotations,the early rice was seeded in middle May and harvested in the late July,and the late rice was seeded in late July and harvested in the early November. In the two annual crop rotation,the rice was seeded in middle June and harvested in the early November.The barley and rice were harvested by hand.The aboveground biomass was an-nually removed with a less than3-cm stubble.Soil sampling and analysisIn each replicated plot,a composite soil sample(pooling soils sampled from five different points)was collected at0–20-cm depth using a soil auger after harvest in November2013.The fresh soil samples were sieved to2mm after removing stones, crop debris,and visible fauna.A part of fresh soil samples was analyzed for MBC and DOC contents,and the other part was air-dried at room temperature and removed inorganic C using dilute HCl before LOC,POC,and total organic C(TOC) analysis.Chloroform-fumigated and chloroform-unfumigated sam-ples were extracted with0.5M K2SO4(Vance et al.1987)and analyzed using an automated TOC analyzer(Analytik Jena, Germany);MBC was calculated by an extraction efficiency coefficient(K EC)value of0.45(Jenkinson et al.2004).DOC was extracted with a soil/water ratio of1:5(g g−1)at25°C;the soil suspension was shaken for0.5h and then centrifuged at 1250g for10min;the supernatant was filtered to pass through 0.45μm(Gong et al.2009).The C concentration in the filtrate was determined using the automated TOC analyzer(Analytik Jena,Germany).LOC was measured following Blair et al. (1995).Soil through a0.25-mm sieve,containing15mg C, was weighed into50-ml centrifuge tubes and treated with 25ml333mM KMnO4.The centrifuge tubes were shaken for6h and centrifuged for5min at815g.The absorbance of the supernatant and standards was read at565nm.The change of KMnO4concentration was used to estimate the amount of C oxidized,assuming that1mM KMnO4is consumed in the oxidation of9mg of C.POC was ana-lyzed following Ouédraogo et al.(2006);100-g soil was dispersed in300ml of distilled water containing three glass bullets and shaken for1h.The dispersed sample was sieved(<53μm).The material remaining on the sieve was dried in a forced-air oven at60°C,weighed after correcting for sand content,and ground to pass a53-μm sieve for C concentration analysis using an elemental an-alyzer(Elementar,Germany).Lastly,TOC concentration was determined by an elemental analyzer(Elementar, Germany)using the air-dried soil.Bulk density was measured using a conventional core method with a volume of100cm3after removing rocks.Soil C storage at0–20-cm depth was calculated on an area basis using TOC content and bulk density(Eq.1).CS¼C TOCÂBDÂ2ð1Þwhere CS is the C storage at0–20-cm depth(Mg C ha−1), C TOC refers to TOC concentration(g kg−1),and BD represents bulk density(g cm−3).Measurements of grain yield,straw,and total shoot biomassIn each plot,grain and straw of each crop were wholly har-vested and air-dried.Then,the grain yield and straw biomass of each crop were calculated separately.The total shoot bio-mass of each crop was the sum of grain yield and straw biomass.Estimates of cumulative C input and SOC sequestration The23-year cumulative C input of pig manure was estimated using Eq.2.CI M¼M fÂ1−W MðÞÂC MÂ23ð2Þwhere CI M is the cumulative manure-C input(Mg C ha−1),M f is the annual fresh pig manure application(22.5Mg ha−1year−1), W M refers to water content of fresh pig manure(68.9%),C M is the C concentration of dry pig manure(197.4g kg−1dry mat-ter),and23represents the number of years.The23-year cumulative root-C input of each plot was esti-mated using Eqs.3–5.CI Root¼ΣBarley RootþΣRice Rootð3ÞΣBarley Root¼B SÂ1−W BðÞÂR B‐R=SÂC BRÂ23ð4ÞΣRice Root¼ER SþLR SðÞÂ10þR SÂ13½Â1−W RðÞÂR R‐R=SÂC RRð5Þwhere CI Root is the cumulative root-C input(Mg C ha−1),ΣBarley Root refers to cumulative barley root-C input(Mg C ha−1),ΣRice Root is the cumulative rice root-C input(Mg C ha−1),B S represents annual barley shoot(grain+straw)bio-mass(Mg C ha−1),W B refers to water content of barley shoot (12%),R B-R/S is the average ratio of root to shoot biomass of barley(17.9%),reported by Yang et al.(2011),Zhang et al. (2011),and Wang et al.(2013),C BR is the C concentration of dried barley root(42%),ER S and LR S are annual shoot(grain +straw)biomass of early rice and late rice(Mg C ha−1)during the first10years,respectively,R S is the annual shoot(grain+ straw)biomass of single cropping rice(Mg C ha−1)during 1991–2013,W R refers to water content of rice shoot(14%), R R-R/S is the average ratio of root to shoot biomass of rice (8.7%),reported by Wang et al.(2006),Zhao(2010),YaoBiol Fertil Soils(2011),and Chu(2013),and C BR is the C concentration of dry barley root(41%).The23-year total C input(manure+root)was estimat-ed using Eq.6.The SOC sequestrations of CK,M,NPK, and MNPK compared with the initial C stock were esti-mated using the Eq.7.The23-year total C outputs of CK, M,NPK,and MNPK treatments were estimated using Eq.8.CI Total¼CI MþCI Rootð6ÞSOC SA=I¼CS Treatment−CS Initialð7ÞCO Total¼CI TotalþCS CK−CS Treatmentð8Þwhere CI Total is the total C input(Mg C ha−1),SOC SA/I is the SOC sequestration amounts compared with the initial C stock (Mg C ha−1),CS Treatment is the SOC stock of different treat-ments(Mg C ha−1),CS Initial is the initial C stock(41.2Mg C ha−1),and CO Total is the total C output(Mg C ha−1).Data analysisStatistical analysis was performed with SPSS13.0(SPSS Inc., Chicago,IL).Multiple comparisons among different fertiliza-tions were analyzed using a one-way analysis of variance (ANOV A)with a Tukey HSD test.Linear relationships were quantified using Pearson’s correlation coefficients. Figures were drawn by ORIGIN8.0(OriginLab Corporation, Northampton,MA).ResultsTOC,bulk density,SOC storage,and fractions Compared with the CK treatment,M,NPK,and MNPK treat-ments significantly increased TOC concentrations after 23years(Fig.1a).The MNPK and M treatments had similar TOC contents,while they were both significantly higher than the TOC content of the NPK treatment.In contrast,M,NPK, and MNPK treatments did not change the bulk densities com-pared with the CK treatment(Fig.1b).Therefore,SOC stocks of the four treated soils had similar patterns as TOC concen-trations(Fig.1c).The impacts of four fertilizations on SOC fractions and their contributions to TOC are shown in Table1.The M, NPK,and MNPK treatments,especially manure amendment, increased the MBC content and its contribution to TOC. Compared with the CK treatment,manure application in-creased the DOC content,but the NPK addition had no impact on the DOC content.The three fertilizations increased the POC content and its contribution to TOC compared with the CK treatment.Cumulative crop biomass and total C inputCompared with the CK treatment,fertilization treatments,par-ticularly chemical fertilizer addition,significantly increased grain yields,straws,and total shoot biomass(combination of yield and straw)(Table2).The estimated23-year total root C i n p u t s i n t o s o i l s c o u l d b e r a n k e d a s f o l l o w: MNPK>NPK>M>CK(Table3).Both the M and MNPK treatments received an external C input of32Mg C ha−1 (Table3).When adding manure-C,the order of cumulative total C inputs(manure+root)was as follows: MNPK>M>NPK>CK(Table3).The total C inputs were pos-itively correlated with the TOC,MBC,DOC,LOC,and POC contents(Table4).SOC sequestration and its response to total C input After23years,the SOC sequestrating amounts of the CK, M,NPK,and MNPK treatments were−3.67,4.45,0.32, Fig.1Total organic C(TOC),bulk density,and soil organic C(SOC)stock of soil subjected to different fertilizer treatments after23-yearcultivation.CK control,M pig manure,NPK chemical NPK fertilizer,MNPK manure combined with chemical NPK fertilizer.Each valuerefers to the average of three replicates,and error bar represents thestandard deviation(SD).Different lowercase letters above the columnsdenote significant differences at P<0.05levelBiol Fertil Soilsand6.45Mg C ha−1,respectively,considering an initial SOC stock of41.2Mg C ha−1(Fig.2a).The annual SOC seques-trating rates of the CK,M,NPK,and MNPK treatments were −0.16,0.19,0.01,and0.28Mg C ha−1year−1,respectively (Fig.2b).The SOC sequestrations were linearly correlated with total C inputs(R2=0.88,P<0.001)(Fig.2c),indicating that(i)the investigated soil had not reached the SOC satura-tion value,(ii)the conversion rate from input-C to SOC was 21%,and(iii)a C input threshold of1.17Mg C ha−1year−1 was needed to maintain the initial SOC level. DiscussionEffects of various fertilizer applications on SOC stock and its fractionsPrior to the experiment,the field had been intensively cultivated for at least30years and subjected to chemical fertilization.This long-term cropping history certainly allowed reaching a relatively steady SOC level. Consequentially,the changes in C input would lead to variations of SOC storage.Therefore,it is reasonable that the CK treatment decreased9%of SOC stock compared with the initial SOC level(Fig.2a)due to the lower residue-C input.Our result agrees with data of subtropical paddy soil in Nanchang,southern China(Zhang et al. 2012),and a sandy loam soil in the North China Plain (a decrease of7%)(Fan et al.2014).By contrast,high manure-C amendment(M and MNPK)raised the SOC storage.On the contrary,the NPK fertilization changed little the SOC stock,confirming results in some subtrop-ical warm and humid soils of southern China(Huang et al.2010;Zhang et al.2012)and a humid rice–berseem soil in subtropics of India(Majumder et al.2008).The changes in the soil MBC,DOC,LOC,and POC con-tents can be influenced by changes in C inputs(Bolinder et al. 1999;Xu et al.2011).Both fertilizer amendments,especially manure application,increased MBC content and its contribu-tion to TOC(Table1),thus confirming what was observed in a subtropical paddy soil in China(Yan et al.2007),subtropical arable soils of India(Rudrappa et al.2006;Mandal et al. 2007),a tropical agricultural soil(Chakraborty et al.2011),a dryland in northwest China(Liu et al.2013),and a sandy loam soil in Henan,China(Zhang et al.2014).It is reasonable that C addition provides substrates for soil microorganisms,thus stimulating their growth(Zhang et al.2014).The significant (R2=0.94,P<0.01)linear correlation between MBC and TOC (Table4)further supports this concept.The DOC,LOC,and POC,considered as active frac-tions of SOC,responded quickly to fertilization,especial-ly to manure amendment(Six et al.2002;Gosling et al. 2013).The DOC is supposed to be derived from rhizodeposition,plant residues,and humus(Kalbitz et al. 2000).Manure application increased DOC content,simi-lar as a soil in subtropics of India(Brar et al.2013),but NPK addition had no impact on DOC content compared with the CK treatment(Table1)and this may suggest that the DOC was derived from manure-C input.The LOC fraction and its contribution to TOC were not affectedTable1Concentrations of soil organic C fractions and the percentages of to total organic C(TOC)Treatments Concentrations Contributions to TOC(%)MBC(mg kg−1)DOC(mg kg−1)LOC(g kg−1)POC(g kg−1)MBC DOC LOC POCCK126.5±4.1c289.5±19.2c 1.96±0.25b 3.35±0.09c0.8±0.1b 1.92±0.21b12.9±1.6a22.2±0.5c M273.6±9.1a409.3±34.3b 2.65±0.22ab 5.57±0.36b 1.5±0.1a 2.16±0.16b14.0±1.3a29.5±1.9ab NPK166.2±18.0b297.2±29.7c 2.08±0.02b 4.80±0.02b 1.0±0.1b 1.74±0.14b12.2±0.3a28.1±0.7b MNPK287.7±9.6a555.1±29.6a 3.35±0.69a 6.35±0.47a 1.5±0.1a 2.81±0.13a17.0±3.6a32.2±2.1aDifferent lowercase letters denote significant differences at P<0.05levelTable223-year cumulative grain yields,straws,and total shoot biomasses Treatments Grain yield(Mg ha−1)Straw(Mg ha−1)Total shoot biomass(Mg ha−1)Barley Rice Barley Rice Barley Rice Barley+riceCK50±9c147±5c54±7c123±6b105±16c269±11b374±27cM65±5b171±10b66±4b147±12ab131±8b318±22ab449±29b NPK75±2ab187±10ab78±3a162±10a153±1ab349±20a502±21ab MNPK84±3a200±11a89±3a169±21a173±5a368±32a541±28aDifferent lowercase letters denote significant differences at P<0.05levelBiol Fertil Soilsby either manure or chemical fertilizer amendment (Table1),similar as a subtropical paddy soil in China (Yan et al.2007).The relatively faster decomposition and turnover rate of LOC than TOC(Six et al.2002) may explain this phenomenon.The POC is mainly com-posed of plant residues at various stages of decomposition (Six et al.2002),and thus,it represents the balance be-tween crop residues input and their output due to decom-position(Gulde et al.2008).Fertilization increased POC concentration and its contribution to TOC(Table1)be-cause of the high residue-C and manure-C input(Table3), thus confirming what was observed in a subtropical paddy soil in China(Yan et al.2007),two humid arable soils in subtropical India(Rudrappa et al.2006;Majumder et al. 2008;Purakayastha et al.2008),and a Black Chernozem soil(Campbell et al.1991)and a Dark Brown Chernozem clay loam soil(Gulde et al.2008)both in Canada.The increase of POC may depend on the higher C input and/or a lower degradation since the fraction may be stabilized.In our study,the SOC and its fractions were correlated,and they were all significantly related to C inputs.The result agrees with what was observed in the thick Black Chernozem at Melfort,Canada(Campbell et al.1991),and a dry land in northwest China(Liu et al.2013).Response of SOC sequestration to C inputWe have shown a linear response of SOC sequestration to fertilization-induced C input(Fig.2c)as already shown (Kong et al.2005;Kundu et al.2007;Zhang et al.2010; Fan et al.2014).This suggests that the investigated soils were not C saturated.The same has been observed in arable soils of subtropical China(Huang et al.2010),Davis,USA (Kong et al.2005),in the Indian Himalayas(Kundu et al. 2007),and in the northern China(Zhang et al.2010). However,despite C inputs similar to others,no relationship was found between SOC sequestration and C input for the cropped soil at Melfort,Canada(Campbell et al.1991),and the soil at Lethbridge in southern Alberta,Canada(Gulde et al.2008).Probably,this depends on the different SOC saturation levels among the compared soils(Six et al.2002; Stewart et al.2009).According to the conceptual SOC model proposed by Six et al.(2002),our studied soil holds a potential for SOC sequestration due to the substantial saturation deficit.Therefore,organic fertilizer amendment is needed to enhance SOC sequestration.The slope of the relationship between SOC sequestra-tion and C input corresponds to the conversion rate from input-C to SOC,which was21%in our soil.This value is comparable with those observed in a subtropical humid paddy soil in Wuchang of southern China(20%) (Zhang et al.2012),a subtropical humid soybean–wheat rotation soil of Indian(19%)(Kundu et al.2007),a hu-mid rice–berseem soil in subtropics of India(24%) (Majumder et al.2008),a humid arable soil in subtropical India(15%)(Purakayastha et al.2008),temperate semi-arid soils of USA(14–21%)(Rasmussen and Collins 1991),and a dry soil in Alberta,Canada(26.8%) (Malhi et al.2011).In contrast,lower conversion rates were found in the agricultural soils of Davis,USA (7.6%)(Kong et al.2005),and the arable soils in the North China Plain(14.1%)(Fan et al.2014).These dif-ferent results probably depend on diverse climatic condi-tions for crop growth,soil microbial activity,and lower decomposition rate under flooded conditions during rice growth.Indeed,the warm and humid climate can acceler-ate the C output due to the decomposition of SOC (Fig.3a)and the ratio of C output to C input(Fig.3b) compared with climatic conditions of Davis,CA(Kong et al.2005),and North China Plain(Fan et al.2014).As a result of the fast rates of C output,equilibrium levels ofTable3Estimates of23-year cumulative total C inputs into soils Treatments Root biomass C input(Mg C ha−1)Composted manure C input(Mg C ha−1)Total C input(Mg C ha−1) Barley RiceCK 6.9±1.0c8.3±0.3b–15.2±1.4dM8.7±0.5b9.8±0.7ab3250.5±1.2bNPK10.1±0.1ab10.7±0.6a–20.8±0.7cMNPK11.4±0.3a11.3±1.0a3254.7±0.7aDifferent lowercase letters denote significant differences at P<0.05levelTable4Pearson’s correlation coefficients(r)between total C input,TOC,MBC,DOC,LOC,and POC(n=12)Total C input TOC MBC DOC LOC POCTotal C input 1.00TOC0.93** 1.00MBC0.99**0.94** 1.00DOC0.89**0.85**0.85** 1.00LOC0.80**0.77**0.76**0.86** 1.00POC0.90**0.96**0.92**0.84**0.70* 1.00*P<0.05,**P<0.01,significant correlationsBiol Fertil SoilsSOC are not easily achieved.Further research should fo-cus on the balance between C input and C output.Our data indicated that the paddy –barley cropping sys-tem must receive a C input of 1.17Mg C ha −1year −1to keep the initial SOC level (Fig.2c ).This threshold of C amendment requirement for maintaining the SOC level is comparable to subtropical humid rice soils in Wangcheng (1.69Mg C ha −1year −1),Jinxian (1.11Mg C ha −1year −1),and Wuchang (0.75Mg C ha −1year −1)in southern China (Zhang et al.2012).In contrast,the threshold of C input to maintain the initial SOC level is lower than that of a subtropical rice soil at Nanchang,southern China (3.61Mg C ha −1year −1)(Zhang et al.2012),a hot humid rice –berseem cropped soil in subtropics of India (3.3Mg C ha −1year −1)(Majumder et al.2008),an arable soil at Davis,CA,under Mediterranean-like climate (3.1Mg C ha −1year −1)(Kong et al.2005),and a sandy loam soil in the North China Plain (2.04Mg C ha −1year −1)(Fan et al.2014).Considering that the residue C input of the NPK treatment (0.9Mg C ha −1year −1)did not change the SOCcontent even after 23-year cultivation,our estimated threshold of C amendment is reasonable.ConclusionsThe rice –barley cropped soil of eastern China had not reached a SOC saturation by considering the 0–20-cm depth,and thus,it holds a great potential for SOC seques-tration.The conversion rate from input-C (root residue-C +manure-C)to SOC was 21%,and a critical C input of 1.17Mg C ha −1year −1was needed for maintaining the initial SOC level.An efficient SOC sequestration mainly depends on the amendment of organic fertilizer but not on chemical fertilization.Furthermore,manure addition also increased the contents of sensitive fractions of SOC such as MBC,DOC,LOC,and POC.Future research should determine the potential difference in conversion rate be-tween the root residue-C andmanure-C.Fig.2SOC sequestration compared with the initial SOC stock (41.2Mg C ha −1)(a ),annual SOC sequestration rate compared with the initial SOC level (b ),and the relationship between SOC sequestration and cumulativeC input (c ).Each value refers to mean±SD (n =3).Different lowercase letters denote significant differences at P <0.05levelFig.3Estimated cumulative C outputs from soils (a )and the ratio of cumulative C output to cumulative C input (b ).Each value refers to mean±SD (n =3).Different lowercase letters denote significant differences at P <0.05levelBiol Fertil Soils。