long-term field fertilization affects soil nitrogen transformations in a rice-wheat-rotation...

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J. Plant Nutr. Soil Sci. 2012, 175, 939–946 DOI: 10.1002/jpln.201200149 939

Long-term field fertilization affects soil nitrogen transformations in arice-wheat-rotation cropping systemJinbo Zhang1, Zucong Cai1*, Wenyan Yang1, Tongbin Zhu1, Yongjie Yu 1, Xiaoyuan Yan2, and Zhongjun Jia2

1 School of Geography Sciences, Nanjing Normal University, Nanjing 210097, China2 State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, 210008, China

AbstractMineralization and nitrification are the key processes of the global N cycle and are primarily dri-ven by microorganisms. However, it remains largely unknown about the consequence of intensi-fied agricultural activity on microbial N transformation in agricultural soils. In this study, the 15N-dilution technique was carried out to investigate the gross mineralization and nitrification in soilsfrom a long-term field fertilization experiment starting from 1988. Phospholipid fatty acids (PLFA)analysis was used to determine soil microbial communities, e.g., biomasses of anaerobic bacter-ial, bacterial, fungi, and actinobacteria. The abundance of ammonia-oxidizing bacteria (AOB)and archaea (AOA) were measured using real-time quantitative polymerase chain reaction. Theresults have demonstrated significant stimulation of gross mineralization in the chemical-fertili-zers treatment (NPK) ([6.53 ± 1.29] mg N kg–1 d–1) and chemical fertilizers–plus–straw treatment(NPK+S1) soils ([8.13 ± 1.68] mg N kg–1 d–1) but not in chemical fertilizers–plus–two times strawtreatment (NPK+S2) soil when compared to the control-treatment (CK) soil ([3.62 ± 0.86] mgN kg–1 d–1). The increase of anaerobic bacterial biomass is up to 6-fold in the NPK+S2 comparedto that in the CK soil ([0.7 ± 0.5] nmol g–1), implying that exceptionally high abundance of anae-robic bacteria may inhibit gross mineralization to some extent. The gross nitrification showsupward trends in the NPK+S1 and NPK+S2 soils. However, it is only significantly higher in theNPK soil ([5.56 ± 0.51] mg N kg–1 d–1) compared to that in the CK soil ([3.70 ± 0.47] mgN kg–1 d–1) (p < 0.05). The AOB abundance increased from (0.28 ± 0.07) × 106 copies (g soil)–1

for the CK treatment to (4.79 ± 1.23) × 106 copies (g soil)–1 for the NPK treatment after the 22-year fertilization. In contrast, the AOA abundance was not significantly different among all treat-ment soils. The changes of AOB were well paralleled by gross nitrification activity (gross nitrifica-tion rate = 0.263 AOB + 0.047 NH�

4 -N + 2.434, R2 = 0.73, p < 0.05), suggesting the predomi-nance of bacterial ammonia oxidation in the fertilized fields.

Key words: 15N dilution technique / gross N transformation / phospholipid fatty acids (PLFA) /quantitative PCR / amoA gene / long-term field fertilization

Accepted May 18, 2012

1 Introduction

It has been estimated that the agroecosystems receive 75%of the reactive N created by human action (100 Tg N y–1)(Galloway et al., 2004). Nitrogen transformation in agriculturalsoils has received considerable attention recently, because ofthe increasing awareness that soil, air, and water quality canbe influenced by agricultural activities. Intensive cropping hasgreatly changed the N cycling in soils, resulting in increasedNO and N2O emissions and other N losses (Ding et al.,2010). Therefore, an appropriate management of the Ncycling is required to improve soil fertility and crop productionas well as to decrease the N2O emission and other N losses.Previous studies from long-term experiments have improvedour knowledge on the effects of various fertilization practiceson the N cycling (i.e., NO and N2O emission) (Meng et al.,2005; Ding et al., 2010). It has been reported that manurecould result in significant increase in gross N mineralization(Sørensen, 2001; Habteselassie et al., 2006). However,Gibbs and Barraclough (1998) reported that the addition of alabile organic matter (OM) to soil could not affect gross N

mineralization, but could markedly increase immobilization inshort-term experiments (29 d). Thus, manure applicationsmay reduce the build-up of NO�

3 and the associated N losses(leaching or gaseous N) compared to mineral fertilizers(Meng et al., 2005). Zhang et al. (2011) found that long-term(17 y) mineral-N-fertilizer application could stimulate both soilgross mineralization and NH�

4 immobilization, which in theOM-treated soils, however, were not statistically differentfrom those in the control (no fertilization), despite the largestconcentrations of soil OM (SOM), labile OM, soil microbialbiomass C, and total soil microbial counts observed in theOM treatment (Zhang et al., 2011). To date, the effects andmechanisms of the long-term mineral- and organic-fertilizermanagement on the N cycle in typical rice–wheat rotationsoils in China remain largely unexplored.

Soil N transformations are directly driven by microbial activity(Martens, 1995). The production and consumption of inor-ganic N is, at least partly, a function of the density and level of

* Correspondence: Dr. Z. C. Cai; e-mail: [email protected]

activity of the soil microbial community and exoenzyme pro-duction (Schimel and Weintraub, 2003). Community-levelphospholipid fatty acid (PLFA) profiles have been found to beuseful in detecting the responses of soil microbial commu-nities to a variety of land uses or disturbances in natural eco-systems (Hedrick et al., 2000; Harris, 2003). Certain markerPLFAs, e.g., cy17:0, cy19:0 were chosen to represent anae-robic bacteria (Vestal and White, 1989) and the PLFAs18:1x9 were used as an index for fungi (Frostegrd et al.,2011), can indicate the relative amounts of certain functionalgroups of organisms in soils and thus help us to understandthe element cycling in soils. Recent findings have suggestedthat the ammonia-oxidizing bacteria (AOB) and archaea(AOA) contribute significantly to the autotrophic oxidation ofammonia to nitrite, which is the first and rate-limiting step ofnitrification (Leininger et al., 2006; Jia and Conrad, 2009).Real-time PCR has been successfully used for the quantifica-tion of AOB and AOA population size in soil (Francis et al.,2005; Chu et al., 2007; Jia and Conrad, 2009). The AOA andAOB are ubiquitously distributed in agricultural soil (Leiningeret al., 2006). Therefore, it is effective to investigate theresponses of abundance and composition of the AOB andAOA community to N-fertilizer treatments for understandingthe mechanisms of soil N cycle in different N-fertilizer treat-ments.

The objective of this study is to characterize the potentialgross mineralization and nitrification rates, and to elucidatethe potential mechanisms of long-term mineral-fertilizer andstraw management which affect the soil N cycle. This is ap-proached by examining the soil microbial communities, theabundance of AOB and AOA community, and the relation-ships between the soil properties and gross mineralization aswell as nitrification rates in a long-term (22 y) field experimentof different fertilizer managements in a typical rice–wheatrotation field in China.

2 Materials and methods

2.1 Long-term fertilization experiment and soilsampling

The long-term fertilization experiment was performed at theChangshu Agro-ecological Experimental Station (Institute ofSoil Science, Chinese Academy of Sciences). The soil type isa Gleyic-Stagnic Anthrosols (WRB-FAO) which was devel-oped from lake sediment (Wushan soil). This experiment wasinitialized in 1988 with four treatments, including control treat-ment (CK), chemical-fertilizers treatment (NPK), chemicalfertilizers–plus–straw treatment (NPK+S1), and chemical fer-tilizers–plus–two times straw treatment (NPK+S2), in tripli-cate plots (4 m × 5 m) separated by brick frames. For theNPK treatment, 180 kg urea-N, 75 kg P (as Ca-superpho-sphate), and 150 kg K (as KCl) per hectare were applied inboth the wheat growing season and the rice growing season.For the NPK+S1 and NPK+S2 treatments, rice straw (appliedduring wheat growing season, average total C: 423 g kg–1,total N: 7.2 g kg–1, C : N ratio: 58.7) or wheat straw (appliedduring rice growing season, average total C: 465 g kg–1, totalN: 4.8 g kg–1, C : N ratio: 96.9) was applied at 2250 kg ha–1

(NPK+S1) and 4500 kg ha–1 (NPK+S2) in each crop season,respectively. None of inorganic and organic fertilizers wasapplied in the CK treatment. Rice–wheat rotation is main-tained in the field, and soil sampling was performed from tri-plicate plots of each treatment during wheat growing season.

Bulk soil (10 cores, each diameter 5 cm) was collected (top0–10 cm) from each plot, passed through a 2 mm sieve, andthen immediately transported to the laboratory in May, 2010.A 50 g subsample of each soil sample was stored at –20°Cfor molecular analysis. A 1000 g subsample of each soil sam-ple was stored at 4°C for gross-mineralization and nitrifica-tion-rates analysis. The rest was air-dried and used for physi-cochemical analysis.

2.2 15N-dilution experiment

Gross N mineralization and nitrification rate were determinedusing the 15N-dilution technique. If the NH�

4 and NO�3 pools

are labeled in parallel treatments, it is possible to determinethe gross mineralization and nitrification rates, respectively,using the 15N-dilution principles (Kirkham and Bartholomew,1954; Stark, 2000). There were two treatments (each withthree replicates): in one, the NH�

4 pool was labeled using(15NH4)2SO4 (10 atom% excess) and in the other, the NO�

3pool was labeled using K15NO3 (10 atom% excess). For eachsoil, a series of 250 mL Erlenmeyer flasks was prepared with20 g of fresh soil (oven-dry basis). A volume of 2 mL of(15NH4)2SO4 or K15NO3 solution was added to each of theflasks at a rate of 30 mg NH�

4 -N (kg soil)–1 or 30 mg NO�3 -N

(kg soil)–1. The soil was adjusted to 50% water-filled porespace and incubated for 24 h at 25°C. The flasks were sealedwith silicone rubber stoppers. The soils were extracted at0.5 h and 24 h after fertilizer application for the determinationof the concentration and isotopic composition of the NH�

4 andNO�

3 . Ammonium and NO�3 were extracted with 2 M KCl at a

soil-to-solution ratio of 1:5 on a mechanical shaker for 60 minat 300 rev min–1 at 25°C.

The isotopic composition of the NH�4 and NO�

3 was measuredusing an automated C/N analyzer isotope-ratio mass spectro-meter (Europa Scientific Integra, UK). The NH�

4 and NO�3

were separated for 15N measurements by distillation with Mgoxide and Devarda’s alloy (Feast and Dennis, 1996; Zhanget al., 2009). The H2SO4 solution containing NH�

4 was thenevaporated to dryness at 65°C in an oven and analyzed for15N abundance.

Rates of N mineralization and nitrification were calculatedusing the equations developed by Kirkham and Bartholomew(1954) (Eq. 1).

F = [(Q1 – Q2) × ln(A1 / A2)] / [t × ln(Q1 / Q2)], (1)

where F is the mineralization or nitrification rate (mg kg–1

d–1), t is the incubation time (d), Q1 is the initial 14+15N pool(mg kg–1), Q2 is the post-incubation 14+15N pool (mg kg–1), A1is the initial atom% 15N excess, and A2 is the post-incubationatom% 15N excess.


940 Zhang, Cai, Yang, Zhu, Yu, Yan, Jia J. Plant Nutr. Soil Sci. 2012, 175, 939–946

2.3 Soil-chemical analysis

All chemical analyses were performed on air-dried soil. ThepH was determined on a 1:2.5 soil/water mixture. Soil organicmatter was analyzed by a wet-digestion with H2SO4-K2Cr2O7,and the total N (TN) was determined by semi-micro-Kjeldahldigestion using Se, CuSO4, and K2SO4 as catalysts (Lu,2000). Soil organic matter was transformed to soil organic C(SOC) by multiplying the Bemmelen index (0.58), which waslater used to calculate the C : N ratio. The inorganic N spe-cies (NH�

4 and NO�3 ) were extracted from fresh soil with 2M

KCl and determined by a continuous-flow analyzer (SA1000,Skalar, The Netherlands). The data of coarse-fraction OM(CFOM) as well as light-fraction OM (LFOM) concentrationwas cited from previous reference (Yan and Wang, 2008).The CFOM was the OM concentration in sand-sized(53–2000 lm) soil separates. The air-dried soil was dis-persed in 5% Na-polyphosphate solution, shaken for 8 h, andpassed through nested sieves of 53 lm using a flow ofdistilled water to ensure separation (Willson et al., 2001). TheLFOM was separated by flotation in a ZnBr2 solution(1.7 g cm–3) before and after aggregate disruption (Comptonand Boone, 2002).

2.4 PLFA analyses

The PLFA profiles were analyzed to determine microbial-community composition. Briefly, lipids were extracted from0.5 g soil samples using the modified Bligh and Dyer method(Bligh and Dyer, 1959; Yao et al., 2006). Each soil samplewas extracted by 15 mL Bligh & Dyer extracting solution(0.1 M citric acid buffer: chloroform: methanol = 0.8:1:2v:v:v). Extracts were separated with chloroform, acetone, andmethanol on a silica solid-phase extraction column (SPE-SI).Nitrogen-gas-dried phospholipids were then hydrolyzed andsaponified with alkaline methanol to generate fatty acidmethyl esters (FAME). The PLFA were determined by MIDISherlock Microbial Identification System (MIDI, Newark, DE,USA). The abundance of individual fatty acid methyl-esterswas expressed as mole percentage. Nomenclature of fattyacids followed that used by Frostegård et al. (1993). i15:0,a15:0, i16:0, 16:1x9, 16:1x7t, 17:0, i17:0, a17:0, cy17:0,18:1x7, and cy19:0 were chosen to represent bacteria(Petersen et al., 2002); cy17:0, cy19:0 were chosen to rep-resent anaerobic bacteria (Vestal and White, 1989). ThePLFAs 18:1x9 were used as an index for fungi (Frostegårdet al., 2011). 10Me16:0, 10Me17:0, 10Me18:0 were chosento represent actinobacteria (Kroppenstedt, 1985).

2.5 Soil DNA extraction and quantitativepolymerase chain reaction

DNA was extracted from ≈ 0.5 g fresh soil (for each replicateof each treatment) using the Fast DNA SPIN kit for soil (MPBiomedicals) following the manufacturer’s instruction. Thequality and quantity of DNA were checked using a NanoDropspectrophotometer (NanoDrop Technologies Inc, Wilmington,DE, USA). Quantitative PCR with three replicates for eachsample was performed to enumerate the copy number ofarchaeal and bacterial amoA genes using primer sets Arch-amoAF/Arch-amoAR (Francis et al., 2005) and amoA-1F/

amoA-2R-GG (Rotthauwe et al., 1997) with a CFX96 OpticalReal-Time Detection System (Bio-Rad Laboratories, Inc. Her-cules, CA), respectively. The qPCR standard was generatedusing plasmid DNA from representative clones containingbacterial or archaeal amoA gene, and a dilution series ofstandard template over five orders of magnitude per assaywas used to optimize qPCR conditions. Blanks were alwaysrun with water as a template instead of soil DNA extract. The25 lL reaction mixture contained 12.5 lL of SYBR Premix ExTaq (TaKaRa Biotech, Dalian, China), 0.25 lM of each pri-mer, and 1.0 lL template. Thermal condition was the sameas those described previously in Jia and Conrad (2009). PCRamplification efficiencies of 95.4% with R2 value of 0.994 and101.2% with R2 value of 0.995 were obtained for archaealand bacterial amoA genes, respectively. Specific amplifica-tion of amoA was checked by confirming a single peak in amelting-curve analysis. The numbers of genes were reportedfor dry-weight soil.

2.6 Statistics

One-way ANOVA with Duncan’s post hoc tests was per-formed to evaluate the differences within datasets at signifi-cance level of 0.05. In order to determine the factors affectingsoil N transformation, we applied a multiple regression analy-sis using SPSS software. A cross-correlation analysis wasfirst applied to exclude multicollinearity issues, before themultiple regression analysis was carried out.

3 Results

3.1 Soil properties

The concentrations of SOM and TN were higher in the NPK,NPK+S1, and NPK+S2 soils than in the CK soil; particularly,they were significantly higher in the NPK+S1 and NPK+S2soils than in the NPK soil (p < 0.05) (Tab. 1). The concentra-tions of CFOM and LFOM in the NPK+S1 and NPK+S2 soilswere significantly higher than those in the CK and NPK soils(p < 0.05). The soil pH decreased significantly after long-termfield fertilizations when compared to the CK treatment (p <0.05). However, there was no substantial difference in C : Nratios between the fertilized and CK soils (Tab. 1).

3.2 Gross mineralization and nitrification rates

The lowest gross mineralization rate ([3.62 ± 0.86] mg N kg–1

d–1) was observed in the CK soil. Surprisingly, gross minerali-zation rates in the NPK+S2 soils were not significantly differ-ent from the CK soil, although the highest gross mineraliza-tion rates were observed in the NPK+S1 soil (Fig. 1). Thegross mineralization rates in the NPK soils were also signifi-cantly higher than in the CK soil. The gross mineralizationrate was significantly and positively correlated with SOM con-tent for the CK, NPK, and NPK+S1 treatments (p < 0.01), butnot for the NPK+S2 treatment (Fig. 2a), despite the fact thatthe highest SOM and labile OM (CFOM and LFOM) concen-trations were observed in NPK+S2 soils.


J. Plant Nutr. Soil Sci. 2012, 175, 939–946 Long-term field fertilization affects soil N transformations 941

The lowest gross nitrification rate ([3.70 ± 0.47] mg N kg–1

d–1) was also observed in the CK treatment (Fig. 1). The high-est gross nitrification rate ([5.56 ± 0.51] mg N kg–1 d–1) wasobserved in the NPK soil, which was significantly higher thanin the CK, NPK+S1, and NPK+S2 soils. However, the grossnitrification rates in NPK+S1 and NPK+S2 soils were notsignificantly different from that in the CK.

3.3 Soil PLFA biomass

The total PLFA (sum of the fatty acids that were identified inthe samples) and bacterial PLFA biomass in the NPK+S1and NPK+S2 soils were significantly higher than that in theCK soil (p < 0.05), and the highest values were found in theNPK+S2 soil (Tab. 2). The total PLFA and bacterial PLFA bio-mass was significantly higher in the NPK+S2 soils than in theNPK soils (p < 0.05), however, there was no significant differ-ence between the NPK+S1 and NPK treatments. The totalPLFA and bacterial PLFA biomass in the NPK treatment were

not significantly different from CK treatment. Anaerobic-bac-terial biomass and the ratio of the anaerobic bacteria to thetotal PLFA increased with the increase in the total PLFA


CK NPK S1 S20

2

4

6

8

10NitrificationMineralization

Gro

ss n

itrifi

catio

n ra

te (m

g kg

-1 d

-1)

Treatments

a

b

a a

CK NPK S1 S20

2

4

6

8

10

Gro

ss m

iner

aliz

atio

n ra

te /

mg

kg-1d-1

Treatments

a

bc

c

ab

Figure 1: Gross mineralization and nitrification rates under differentfertilization treatments. CK: no fertilization control, NPK: NPK mineralfertilizers amendment, NPK+S1: combined NPK mineral fertilizer and2250 kg ha–1 straw amendment each crop season, NPK+S2:combined NPK mineral fertilizer and 4500 kg ha–1 straw amendmenteach crop season. The difference in values with same letter in thesame figure was not significant (p < 0.05).

Table 1: Soil properties from four field treatments (mean ± S.D.).

Treatment1 pH SOM2

/ g kg–1Total N/ g kg–1

C : N CFOM2

/ g kg–1LFOM2

/ g kg–1

CK 7.5 ± 0.2b 34.0 ± 1.9a 2.1 ± 0.1a 10.0 ± 1.0a 9.7a 1.6a

NPK 7.0 ± 0.3a 37.1 ± 1.3b 2.4 ± 0.04b 9.6 ± 0.7a 10.2a 1.6a

NPK+S1 6.9 ± 0.2a 43.8 ± 0.3c 2.5 ± 0.02bc 10.6 ± 0.8a 11.2b 2.1b

NPK+S2 6.9 ± 0.2a 45.2 ± 1.3c 2.6 ± 0.2c 10.7 ± 1.1a 13.1c 2.1b

CV / % 4.4 12.4 8.2 8.8 13.6 15.6

1 CK, no fertilization control; NPK, NPK mineral fertilizers amendment; NPK+S1, combined NPK mineral fertilizer and 2250 kg ha–1 strawamendment each crop season; NPK+S2, combined NPK mineral fertilizer and 4500 kg ha–1 straw amendment each crop season. CV is thecoefficient of variation.2 SOM, soil organic matter; CFOM, coarse-fraction OM; LFOM, light-fraction OM.The same letter in the same column shows that the difference is not significant. Data for CFOM-C and LFOM-C is cited from Yan and Wang(2008).

30 35 40 45 504

6

8

10 CK, NPK, NPK+S1 treatments NPK+S2 treatment

Gro

ss m

iner

aliz

atio

n ra

te /

mg

kg-1 d

-1

Soil organic matter / g kg-1

R2=0.62p<0.01

a

10 15 20 25 30 354

6

8

10

Gro

ss m

iner

aliz

atio

n ra

te /

mg

kg-1 d

-1

Bacterial PLFA biomass / nmol g-1

R2=0.48p<0.05

b

Figure 2: Relationship between gross mineralization rate and soilorganic matter (SOM) (a), and the bacterial PLFA biomass (b). CK:no fertilization control, NPK: NPK mineral fertilizers amendment,NPK+S1: combined NPK mineral fertilizer and 2250 kg ha–1 strawamendment each crop season, NPK+S2: combined NPK mineralfertilizer and 4500 kg ha–1 straw amendment each crop season.


(Tab. 2). An anaerobic-bacterial biomass was up to 6-foldhigher in the NPK+S2 soil compared to that in the CK soil(Tab. 2). The ratio of the anaerobic bacteria to the total PLFAin the NPK+S2 soil was significantly higher than in the CK,NPK, and NPK+S1 soils (p < 0.01), however, no significantdifference was observed among CK, NPK, and NPK+S1 soils(Tab. 2). There were some changes in the ratios of thebacteria, fungi, and actinomycetes to the total PLFA amongthe different treatments, but these were not statisticallysignificant (Tab. 2). The gross mineralization rate increasedwith the increases of the bacterial PLFA biomass in the CK,NPK, and NPK+S1 treatments (p < 0.05) (Fig. 2b). Althoughthe highest total PLFA and bacterial PLFA biomass wereobserved in the NPK+S2 soils, the gross mineralizationrates in the NPK+S2 soils were similar with those in theCK soil.

3.4 Soil AOB and AOA abundance

The bacterial amoA gene (AOB) increased from (0.28 ± 0.07)× 106 copies (g soil)–1 for the CK treatment to (4.79 ± 1.23) ×

106 copies (g soil)–1 for the NPK treatment after the 22-y ferti-lization (Fig. 3). Similarly copies of AOB for NPK+S1 andNPK+S2 with (3.85 ± 0.79) × 106 and (3.68 ± 1.10) × 106

copies (g soil)–1, were also higher than AOB in CK. In con-trast, the archaeal amoA gene (AOA) abundance was not sig-nificantly different among the CK ([3.05 ± 0.63] × 106 copies[g soil]–1), NPK ([4.47 ± 1.14] × 106 copies [g soil]–1),NPK+S1 ([3.50 ± 0.59] × 106 copies [g soil]–1), and NPK+S2([3.21 ± 0.55] × 106 copies [g soil]–1) soils. It was interestingto note that in the CK soil plots, the ratio of the archaeal tothe bacterial amoA gene copy number was 13.3, while alower ratio was observed for the NPK (0.93), NPK+S1 (0.91),and NPK+S2 (0.87) soils. The gross nitrification rates in-creased with the increase of AOB abundance (p < 0.01,Fig. 4a). It seems that there was a linear relationship betweengross nitrification rate and AOA, but these were not statisti-cally significant (Fig. 4b). The multiple regression analysisshowed that the gross nitrification rates were rather corre-lated with the bacterial than the archaeal amoA gene copynumber (AOB) and NH�

4 -N concentrations (gross nitrificationrate = 0.263 AOB + 0.047 NH�

4 -N + 2.434, R2 = 0.73,p < 0.05).


Table 2: Soil microbial community estimated as the total extractable phospholipid fatty acids (PLFA) (mean ± S.D.).

Treatment1 Total PLFA/ nmol g–1

Bacteria/ nmol g–1

Fungi/ nmol g–1

Actinobac-teria/ nmol g–1

Anaerobicbacteria/ nmol g–1

B : T2

/ %F : T/ %

Ac : T/ %

An : T/ %

CK 15.6 ± 4.0a 12.2 ± 2.7a 2.1 ± 0.5a 1.3 ± 0.7a 0.7 ± 0.5a 78.6 ± 2.6 a 13.5 ± 0.1 a 7.9 ± 2.5 a 4.1 ± 1.9a

NPK 23.6 ± 6.1ab 18.1 ± 3.6ab 3.6 ± 0.4bc 1.9 ± 1.6a 0.8 ± 0.5a 76.8 ± 3.6 a 15.7 ± 2.4 a 7.5 ± 5.9 a 3.7 ± 1.7a

NPK+S1 25.9 ± 0.8b 19.9 ± 0.8b 3.1 ± 0.1b 2.9 ± 0.1a 1.1 ± 0.6a 77.0 ± 0.7 a 11.9 ± 0.7 a 11.1 ± 0.1 a 4.4 ± 0.9a

NPK+S2 34.9 ± 6.7c 27.5 ± 5.8c 4.0 ± 0.5c 3.4 ± 0.4b 4.9 ± 0.3c 78.6 ± 1.5 a 11.6 ± 0.7 a 9.8 ± 0.8 a 14.7 ± 3.0c

CV / % 34.8 33.6 26.3 48.0 98.8 2.8 15.1 34.4 32.5

1 CK, no fertilization control; NPK, NPK mineral fertilizers amendment; NPK+S1, combined NPK mineral fertilizer and 2250 kg ha–1 strawamendment each crop season; NPK+S2, combined NPK mineral fertilizer and 4500 kg ha–1 straw amendment each crop season. CV is thecoefficient of variation.2 An : T, the ratio of anaerobic bacteria biomass to total PLFA; B : T, the ratio of bacteria to total PLFA; F : T, the ratio of fungi to total PLFA; Ac : T,the ratio of actinobacteria to total PLFA. The same letter in the same column shows that the difference was not significant.

CK NPK NPK+S1 NPK+S20

2

4

6

8

amoA

cop

y nu

mbe

r / 1

06 (g

-1so

il)

Treatments

AOB AOA

a

b

b b

A

A

AA

Figure 3: Archaeal (AOA) and bacterial (AOB) amoA gene copynumber under different fertilization treatments. Difference in valueswith same letter in the same amoA gene (AOA: capital letter, AOB:lowercase) was not significant (p < 0.05).

0 1 2 3 4 5 6 73

4

5

6

7

R2=0.52p<0.01

AOA copy number / 106 (g -1 soil)AOB copy number / 106 (g -1 soil)

Gro

ss n

itrifi

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n ra

te /

mg

kg-1 d

-1

Gro

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te /

mg

kg-1 d

-1

a

0 1 2 3 4 5 6 73

4

5

6

7 b

R2=0.21p>0.05

Figure 4: Relationship between nitrification and AOB (a) and AOA (b)amoA gene copy number.


4 Discussion

4.1 Effects of long-term field fertilization on grossmineralization

Our results suggest that long-term field fertilization stimulatessoil gross N mineralization, except two times of OM-applica-tion treatment (NPK+S2). This is in line with previous reportsthat N additions stimulate mineralization particularly whenthey are accompanied by OM additions, such as in manure orcomposts (Hatch et al., 2000; Burger and Jackson, 2003;Hall and Matson, 2003). Gross N mineralization increaseswith the increase of SOM and bacterial PLFA biomass in theCK, NPK, and NPK+S1 treatments, implying that the chang-es of gross mineralization under different fertilization regimesmay be due to the shifts in the concentration and compositionof SOM and the amounts of microbes. However, it was note-worthy that gross mineralization rates in the NPK+S2 soilwere significantly lower than that in the NPK+S1 soil but notsignificantly different from those in the CK and NPK treat-ments, although the highest concentrations of SOM, labileOM, and the amounts of microbes were observed in theNPK+S2 soil.

Previous studies showed that the ratio of macroaggregates(250–2000 lm) fraction (> 20%) and OM content in macroag-gregates (12.17 g kg–1) in the long-term organic-fertilizer-amendments soils were significantly higher than in CK (< 5%and 6.63 g kg–1, respectively) and NPK (< 10% and11.68 g kg–1, respectively) treatments (Yu et al., 2012). Prob-ably SOM can be more protected physically in the NPK+S2soil than the other treatment soils. In general, outer surfacesof soil aggregates tend to sustain oxidizing conditions, where-as interior regions are more reducing (Tokunaga et al., 2001).In addition, higher SOM can stimulate the consumption of O2in soil pore space in the NPK+S2 soil. Therefore, the largeamount of organic-fertilizer amendments can increase theanaerobic microsites in the soils. Previous studies revealedthat distribution of microorganisms could change dependingon the position in the soil aggregates. In this study, we foundthe higher anaerobic-bacteria biomass and the ratio of theanaerobic bacteria to the total PLFA in the NPK+S2 treatment(p < 0.01), compared with the other treatment soils, mainlydue to increase the anaerobic microsites in bulk soil ormacroaggregates. It has been shown that OM decompositionis slower under anaerobic condition than aerobic conditions(Vinten et al., 2002). Therefore, the low mineralization rate inthe NPK+S2 treatments may be due to the protection of soilaggregates, the development of anaerobic microsites, speci-fic microbial communities (i.e., high ratio of anaerobic bac-teria to total bacteria), and their interactions.

4.2 Effect of long-term field fertilization on grossnitrification

The bacterial amoA genes, ranging from (0.28 ± 0.07) × 106

copies g–1 (CK) to (4.79 ± 1.23) × 106 copies g–1 in the pre-sent investigation, were similar to the observations in anotherlong-term fertilization experiments in China (bacterial amoAgenes, ranging from 0.12 × 106 (CK) to 2.79 × 106 copies g–1

(mineral-N-application treatment) (Shen et al., 2008). The

significant increase in gross nitrification after long-termmineral-N supply in the NPK soils demonstrates that this Ntransformation is very sensitive to any change in mineral-Nsupply (Schimel and Bennett, 2004). In arable soils, mostammonia oxidation is carried out by autotrophic nitrification(Barraclough and Puri, 1995). The drastic changes in theAOB abundance and the significant correlation betweenbacterial amoA gene abundance and gross nitrification rate(p < 0.01) together suggest the AOB play important role inammonia oxidation. The bacterial amoA gene copy numbersin the NPK treatment are significantly higher than those in theCK treatment in our study, indicating the mineral-N inputcould be the key factor that controls the AOB abundance inthe natural soil environment. However, the gross nitrificationrate in the NPK+S1 and NPK+S2 plots does not show signifi-cant difference from that in the CK soils, but is significantlylower than that in the NPK soil (Fig. 1). The long-termmineral-N application could simulate AOB abundance. How-ever, the increasing bioavailability of C in the applied straw-treatments (NPK+S1 and NPK+S2) soils may promote thegrowth of heterotrophic bacteria. The growth of heterotrophicbacteria can possibly outcompete the AOB growth and thegross nitrification activity (Fauci and Dick, 1994; Shi and Nor-ton, 2000). However, we cannot exclude the possibility thatthe AOA might have contributed to soil nitrification, althoughthe AOA abundance remains stable after 22-year field fertili-zation and significant relationship is not found between AOAabundance and gross nitrification rates. Nevertheless, ourresults demonstrate that the AOA is unlikely to be the key dri-ver for ammonia oxidation.

5 Conclusion

Our results demonstrate that that long-term repeated applica-tions of chemical fertilizers or chemical fertilizers plus lowamount of straw (e.g., 2250 kg ha–1 in this study) could signifi-cantly stimulate gross mineralization, but not in chemical ferti-lizers–plus–two times straw treatment (e.g., 4500 kg ha–1),when compared to the control treatment. Long-term chemi-cal-fertilizers application stimulated gross nitrification, how-ever, chemical fertilizers–plus–straw treatments did not affectthe ammonia oxidation, when compared to the control treat-ment. Therefore, a combination of mineral- and organic-Ntreatment had the lowest effect on potential NO�

3 leachinglosses and can be recommended in this rice–wheat rotationcropping system.

Acknowledgments

This work is founded by Projects of National Natural ScienceFoundation of China (40830531, 41101209 and 40921061),Natural Science Foundation of Jiangsu Province (BK2010611,BK20082282), and the Priority Academic Program Develop-ment of Jiangsu Higher Education Institutions.

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