Effect of orchard age on soil nitrogen transformation in subtropical China and implications

J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 4 (2 0 1 5) 10 – 1 9

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ScienceDirect www.journals.elsevier.com/journal-of-environmental-sciences

Effect of orchard age on soil nitrogen transformation in subtropical China and implications Yushu Zhang1,2,3 , Jinbo Zhang1,3,4,5 , Tongbin Zhu1,3 , Christoph Müller6 , Zucong Cai1,3,4,5,⁎ 1. School of Geography Sciences, Nanjing Normal University, Nanjing 210023, China. E-mail: [email protected] 2. Institute of Soil and Fertilizer, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China 3. Jiangsu Provincial Key Laboratory of Materials Cycling and Pollution Control, Nanjing 210023, China 4. Jiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application, Nanjing 210023, China 5. Key Laboratory of Virtual Geographical Environment, Ministry of Education, Nanjing Normal University, Nanjing 210023, China 6. Department of Plant Ecology, Justus-Liebig University Giessen, 35392 Giessen, Germany

AR TIC LE I N FO

ABS TR ACT

Article history:

A better understanding of nitrogen transformation in soils could reveal the capacity for

Received 16 October 2014

biological inorganic N supply and improve the efficiency of N fertilizers. In this study, a 15N

Revised 18 January 2015

tracing study was carried out to investigate the effects of converting woodland to orchard, and

Accepted 6 March 2015

orchard age on the gross rates of N transformation occurring simultaneously in subtropical

Available online 29 April 2015

soils in Eastern China. The results showed that inorganic N supply rate was remained constant with soil organic C and N contents increased after converting woodland into citrus orchard

Keywords: 15

N tracing technique

and with increasing orchard age. This phenomenon was most probably due to the increase in the turnover time of recalcitrant organic-N, which increased with decreasing soil pH along

Gross rates of

with increasing orchard age significantly. The amoA gene copy numbers of both archaeal and

nitrogen transformation

bacterial were stimulated by orchard planting and increased with increasing orchard age. The

Subtropical orchard soil

nitrification capacity (defined as the ratio of gross rate of nitrification to total gross rate of mineralization) increased following the Michaelis–Menten equation, sharply in the first 10 years after woodland conversion to orchard, and increased continuously but much more slowly till 30 years. Due to the increase in nitrification capacity and unchanged NO−3 consumption, the dominance of ammonium in inorganic N in woodland soil was shifted to nitrate dominance in orchard soils. These results indicated that the risk of NO−3 loss was expected to increase and the amount of N needed from fertilizers for fruit growth did not change although soil organic N accumulated with orchard age. © 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Introduction Orchards are an important agro-ecosystem that occupy > 5 million ha in subtropical regions of China. To achieve high yields and improve market quality of fruit, high rates of inorganic and organic fertilizers are commonly applied to orchards every year. Lu et al. (2012a) surveyed of 916 orchards

in China finding that N application rates averaged 588 kg N/ha. Not surprisingly, high N application rates not only affect soil physical, chemical and biological properties, but also lead to changes in soil N transformation dynamics (Zaman et al., 1999; Lu et al., 2012b; Zhang et al., 2013a), resulting in environmental problems (e.g. N2O emissions and NO−3 leaching and runoff from soil).

⁎Corresponding author. E-mail: [email protected] (Zucong Cai).

http://dx.doi.org/10.1016/j.jes.2015.03.005 1001-0742/© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

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It has been demonstrated that nitrification activity in the natural forest soils in humid subtropical regions of China may be weak or even absent and that NO−3 immobilization may be dominance (Zhang et al., 2011). As a consequence, the inorganic N in these soils is dominated by NH+4, thus effectively preventing N losses through NO−3 leaching and runoff and retaining inorganic N in soils (Zhang et al., 2013b). However, when forestlands are converted to agricultural lands, nitrification is stimulated and microbial immobilization of NO−3 is substantially suppressed (Yang et al., 2008; Zhang et al., 2013a). Therefore, NO−3 dominance in inorganic N in agricultural soils and the mechanism of N retention in humid subtropical soils is destroyed (Yang et al., 2010; Han et al., 2012; Zhang et al., 2013a). It has been observed that management practices, such as fertilization, stimulate the activity of nitrifying bacteria (e.g. archaeal (AOA) and bacterial (AOB)) in the agricultural soils, thus increasing the production of NO−3 (Chu et al., 2008; Shen et al., 2008; Lu et al., 2012b; Zhang et al., 2012a). However, the exact process by which the mechanisms for retaining inorganic N in humid subtropical soils are removed after converting natural forestland to agricultural land is not known. Previous studies have found that soil organic carbon (SOC) and total N (TN) contents increased substantially with orchard age due to amendments with high rates of organic fertilizer in subtropical China (Guo et al., 2010a; Zhong et al., 2011; Zhang et al., 2012c). However, our inquiries found that local fruit farmers held the view that the demand for N fertilizers did not change with increasing age of citrus orchards. Thus, they do not reduce chemical N fertilizer application rates with increasing orchard age, despite SOC and TN accumulating. The majority of soil N is bound in organic forms which is unavailable directly to plants and only small proportion of soil N is in an inorganic forms (mainly as NH+4 and NO−3) and directly available to plants (Bonito et al., 2003). Organic N mineralization is the primary process transforming organic N into inorganic N (Müller et al., 2004; Habteselassie et al., 2006). It has been widely accepted that the mineralization rate usually increases with an increase in total soil organic N content (Rasmussen et al., 1998; Vervaet et al., 2002; Yan et al., 2008). It can be inferred that the rate of biological inorganic N supply will increase with soil organic N accumulation. In order to advise fruit farmers in the optimal N fertilizer, confirming the role of organic matter in supplying N is very important. The objective of this study is to understand how the mechanisms for retaining inorganic N in humid subtropical soils alter after converting natural forestland to agricultural land, and the changes in potential biological inorganic N supply in orchard soils. This is achieved by investigating the gross rates of N transformation in citrus orchard soils of differing ages, using a 15 N tracing study, and adjacent woodland soils as a control.

1. Materials and methods 1.1. Soil samples

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approximately 1700 mm and the mean annual air temperature is 20.3°C. Native vegetation consists of subtropical evergreen forest. Five sites including four citrus orchards, 2, 10, 20, and 30 years old (hereafter referred to as Orch-2, Orch-10, Orch-20 and Orch-30, respectively) and the adjacent woodland, were selected. These sites had similar soil types and management and differed due to the time of land use as an orchard. All sites had similar slope (16°–20°) and aspect (33.0°–67.0°). The soils within these sites were classified as Ultisols (US Soil Taxonomy) and had developed from Granite. All selected citrus orchards sites had been converted from similar secondary woodland dominated by Castanopsis, Lithocarpus and Cyclobalanopsis. The management of the citrus orchards followed standard local practice. Briefly, in the initial stages of woodland conversion to citrus orchard, almost all vegetation was removed and soil was disturbed (surface soil was removed due to conversion slope land to terrace). Generally, organic fertilizers are applied in February and ammonium sulfate or compound fertilizers N are applied in February, May, August and September. Within the first seven years after woodland conversion to citrus orchard, N and organic fertilizers were applied yearly at 300–500 kg/ha and 2000–4000 kg/ha (e.g. pig manure, poultry manure, and cattle manure), respectively. While, after seven years of citrus cultivation, N and organic fertilizers were applied yearly at 500–600 kg/ha and 4000–6000 kg/ha, respectively. To avoid the effect of fertilization, soil samples were collected in December 2012. For each orchard age, three orchards and adjacent woodland were sampled (Fig. 1). At each sample site, six fruit trees were randomly selected, 36 soil cores were randomly collected from 0 to 20 cm under the crowns of these fruit trees after first removing litter. The soil from the 36 soil cores was mixed as a one sample. The fresh soil was passed through a 2-mm sieve, and was then split into two subsamples. One subsample was stored at 4°C for the incubation studies within two weeks and the other was air-dried for analysis of soil properties as described below.

1.2. 15N tracing experiment A 15N tracing experiment following the procedure of Zhang et al. (2011), was performed. There were two NH4NO3 treatments (each with three replicates): one labeled with 15NH4NO3 (9.86 atom% excess) and the other with NH15 4 NO3 (9.82 atom% excess). A series of 250-mL conical flasks was prepared for each soil and each flask contained 30 g of fresh soil (oven-dry basis). One microliter of 15NH4NO3 or NH15 4 NO3 solution was added to each of the flasks at a rate of 60 μg N/g soil (30 μg NH+4-N/g soil and 30 μg NO−3-N/g soil). The soil was adjusted to 60% water holding capacity and incubated at 25°C. The conical flasks were sealed with silicone rubber stoppers which were removed for 1 hr every two days in order to aerate the soil. Three conical flasks from each treatment were randomly selected and the soils were extracted with 2 mol/L KCl at 0.5, 48, 96, and 144 hr after the 15N labeled solutions were added, in order to determinate the concentrations of the NH+4 and NO−3 and their 15N enrichments.

1.3. Analyses The study sites were located in the Yongchun County, Fujian Province, China, which is characterized by a typical subtropical monsoon climate. Mean annual precipitation at the study site is

Soil properties were determined using the methods of Soil Agro-Chemical Analysis procedures (Lu, 2000). Soil pH was

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25°24′N 25°30′N 25°23′N 25°20′N

25°22′N

25°10′N 117°40′E 117°50′E 118°0′E

118°10′E 118°20′E 118°30′E

118°19′E

118°20′E

118°21′E

Fig. 1 – Location of sample sites where nos. 1–3 were located at woodlands, nos. 4–6 at the citrus orchards of 2 years age (Orch-2), nos. 7–9 at the orchards of 10 years age (Orch-10), nos. 10–12 at the orchards of 20 years age (Orch-20), and nos. 13–15 at the orchards of 30 years age (Orch-30).

measured using a DMP-2 mV/pH detector (Quark Ltd., Nanjing, China) at a 1:5 (V/V) soil to water ratio. SOC and total N were analyzed by wet-digestion with H2SO4–K2Cr2O7 and semi-micro Kjeldahl digestion using Se, CuSO4 and K2SO4 as catalysts, respectively. The soil NH+4 and NO−3 contents were determined by extracting with 2 mol/L KCl at a soil/solution ratio of 1:5 and shaking 60 min at 300 r/min at 25°C with a mechanical shaker. After filtering through a filter paper, the extracts were analyzed with a continuous-flow analyzer (Skalar, Breda, Netherlands). Soil labile organic N (Nlab) and recalcitrant organic N (Nrec) were determined using physical fractions in a method described by Zhu et al. (2011) and Meijboom et al. (1995). Briefly, mineral N of fresh sampled soil was extracted using 2 mol/L KCl at a soil/ solution ratio of 1:5, the KCl-extracted soil was wet sieved over a 150 μm sieve. Clay, silt and fine soil particles were removed using deionized water till the clear water flowed out of the sieve. Material remaining on the sieve was decanted into a bucket where it was swirled in 1 L of deionized water. The dispersed material (light fraction) was decanted into a fractionation sieve and washed with deionized water again. After several cycles, the fraction retained at the top of fractionation sieve was dried at 60°C for 24 hr and N content of this fraction was defined as the Nlab pool. Recalcitrant organic N (Nrec) was calculated by total N subtracted mineral N and Nlab. The 15N abundance of NH+4 and NO−3 pools was analyzed with an automated C/N analyzer isotope ratio mass spectrometer (IRMS 20–22, SerCon, Grewe, UK). The NH+4 and NO−3 were separated with following: the KCl extract was steam-distilled with MgO to separate NH+4 from inorganic N on a steam distillation system, thereafter Devarda's alloy was added to the sample in the flask to reduce NO−3 into NH+4 and distilled again to liberate NH+4 reduced from NO−3 (Lu, 2000). The liberated NH3 was trapped in a conical flask using boric acid solution (0.32 mol/L) and converted to (NH4)2SO4 using a 0.02 mol/L H2SO4 solution. The ensuing (NH4)2SO4 solution was then evaporated to dryness in an oven (65°C) before being analyzed for 15N abundance. The recovery of NH+4 and NO−3 with this process was more than 99% and 95%, respectively (Zhang et al., 2011). DNA was extracted from 0.25 g fresh soil using the PowerSoil® DNA Isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) following the manufacturer's instruction. The quantity and quality of DNA were checked by NanoDrop spectrophotometer

(NanoDrop Technologies Inc, Wilmington, DE, USA). Quantitative PCR was used to enumerate the copy number of bacterial and archaeal amoA genes using primer sets amoA-1F/amoA-2R-GG (Rotthauwe et al., 1997) and ArchamoAF/Arch-amoAR (Francis et al., 2005) with a CFX96 Optical Real-Time Detection System (Bio-Rad Laboratories, Inc., CA, USA), respectively. The qPCR standard was generated by plasmid DNA from representative clones containing bacterial or archaeal amoA gene. The 20.0 μL reaction mixture contained 10.0 μL of SYBR Premix Ex Taq (TaKaRa Biotech, Dalian, China), 0.5 μL of each primer, and 1.0 μL template. Thermal condition of quantitative PCR for archaeal amoA genes referred to Francis et al. (2005), following protocol: 95°C for 3 min, 40 cycles consisting of 95°C for 30 sec, 55°C for 30 sec, and then 72°C for 30 sec. Bacterial amoA genes using the thermal condition referred to Rotthauwe et al. (1997). Briefly, 3 min at 95°C, then 40 cycles consisting of 10 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C and 5 sec at 80°C. Fluorescence was detected at the last stage of each cycle. Specific amplification of amoA was checked by confirming a single peak in a melting-curve analysis. Copy numbers of genes are reported per dry-weight of soil.

1.4. 15N tracing model The gross N transformations occurring simultaneously in the soil were quantified with a process based 15N tracing model (Müller et al., 2007). The model includes the mineralization of recalcitrant organic-N (MNrec) and labile organic-N (MNlab) to NH+4; immobilization of NH+4 to labile organic-N (INH4_Nlab) and to recalcitrant organic-N (INH4_Nrec); release of adsorbed NH+4 (RNH4) and adsorption of NH+4 on cation exchange sites (ANH4); oxidation of NH+4 (ONH4) and recalcitrant organic-N to NO−3 (ONrec); and immobilization of NO−3 to recalcitrant organic-N (INO3) and dissimilatory NO−3 reduction to NH+4 (DNO3). The gross N transformation rates are calculated by simultaneously optimizing the kinetic parameters for the various N transformations while minimizing the misfit between modeled and actual 15 N enrichments and concentrations of NH+4 and NO−3 observed in the experiment (Rütting et al., 2008). Initial concentrations of the 14 N and 15N pool size were determined by the concentrations of NH+4 and NO−3 at time zero through back-extrapolation of data at t = 0.5 hr and t = 48 hr (Müller et al., 2004).

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1.5. Calculation and statistical analyses Averages and standard deviations of model parameter were calculated from the probability density function of each parameter using the optimization procedure (Müller et al., 2007). To identify adequate iteration numbers, each analysis run was carried out with three parallel sequences. Based on the kinetic settings and the final parameters, gross N transformation rates were calculated over a 144-hr period. The net nitrification rate (NN) is calculated by Eq. (1):
ð1Þ NN ¼ ðONH4 þ ONrec Þ− I NO3 þ DNO3 The net rate of organic N transforming into inorganic N is taken as an index of biological inorganic N supply rate (NS), which is calculated by Eq. (2):

NS ¼ M Nrec þ M Nlab þ ONrec − I NH4 Nrec þ I NH4 Nlab þ INO3 : ð2Þ Turnover time of organic N (TT) in units of ‘day’ is used to indicate the decomposability of organic N into inorganic N, which is calculated by Eqs. (3)-(4): TTNrec ¼ Nrec =MNrec

ð3Þ

TTNlab ¼ Nlab =M Nlab

ð4Þ

where, TTNrec and TTNlab refer to the turnover time of recalcitrant organic-N and labile organic-N, respectively; and Nrec and Nlab are the contents of recalcitrant organic-N and labile organic-N, respectively. One-way ANOVA was employed to examine the differences in soil properties, gross rates and net rates between the woodland soil and orchard soils of different ages. When the difference was significant at p < 0.05, Duncan's test was used to compare the means of treatments. Pearson correlation coefficient analysis and regression analysis were also performed to explore the relationship between the variables. The data from each site were used for Pearson correlation analysis and regression analysis unless otherwise stated.

2. Results 2.1. Soil properties SOC and TN contents were higher in the orchard soils than in the woodland and increased with orchard age (p < 0.01) with a relative stable C/N ratio (Table 1). Both labile organic-N (Nlab) and recalcitrant organic-N (Nrec) contents increased linearly with increasing total organic N content (p < 0.01 for both Nlab and Nrec, Fig. 2). The regression slopes showed that the increased total N was allocated mainly in the Nrec fraction (76%) and secondly in the Nlab fraction (23%), respectively. Correlation analysis showed that both Nrec and Nlab were negatively correlated with soil pH (r = −0.636, p < 0.05 for Nrec and r = −0.707, p < 0.01 for Nlab). Soil NO−3-N content was higher in orchard soils than in woodland, but the difference was not significant at p < 0.05. However, NH+4-N contents were significantly lower in Orch-10, Orch-20 and Orch-30 than in the woodland soil and Orch-2

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( p < 0.05, Table 1). The nitrate ratio (defined as the ratio of NO−3 in the total inorganic N) showed a tendency to increase with increasing orchard age and the difference was significant between woodland soil and orchard soils (p < 0.05) and between Orch-2 and Orch-30 (p < 0.05). The nitrate ratio indicated that woodland soil was dominated by NH+4 (the ratio was <0.5), while the orchard soils were dominated by NO−3 in inorganic N pool (the ratio was > 0.5). There was a trend for the soil pH to decrease after conversion from woodland (5.19 ± 0.09) into orchard and with orchard age, although the differences were not significant due to the large variations in the orchard at the same age (p = 0.079, Table 1).

2.2. Soil AOA and AOB abundance Fig. 3 shows that the archaeal amoA gene copy numbers (AOA) in woodland soil was 1.33 × 106 copies/g soil, lower than that in orchard soil (from 6.31 × 106 to 3.22 × 108 copies/g soil). Archaeal amoA gene copies increased after conversion to orchard at 2 years old and this difference increased further by 10 years age, while no difference due to age after 10 years (p < 0.05). The copy numbers of bacterial amoA gene were lower in woodland soils than that in orchard soil with no significant difference due to orchard age.

2.3. NH+4 production and consumption Using the 15N tracing model, gross N transformation rates were calculated (Table 2). The results showed that NH+4 production rates were dominated by gross rates of mineralization of recalcitrant organic N (MNrec) and of labile organic N (MNlab ), and the contribution of dissimilatory NO−3 reduction to NH+4 (DNO3 ) was negligible. There were no significant differences in MNrec between woodland soils and orchard soils and no difference due to orchard age, although MNrec was much smaller in Orch-20 and Orch-30 (Table 2). There was a significant negative relationship between Nrec and MNrec ( p < 0.01, Fig. 4). MNlab were significantly higher in the orchard soils than that in the woodland soil (Table 2) and increased significantly with increasing Nlab ( p < 0.05, Fig. 4). There was, at certain extent, an offset between MNrec and MNlab (p = 0.093), the total gross rate of organic N mineralization was not different significantly between woodland and orchard soils and among the orchard soils. There was an exponential relationship between the turnover time of recalcitrant (TTNrec) and soil pH (p < 0.01, Fig. 5a), but the turnover time of labile organic N(TTNlab) was not correlated with soil pH significantly (p > 0.05, Fig. 5b). Ammonium is consumed by the processes of immobilization into organic N (INO4_Nrec and INO4_Nlab) and oxidation into NO−3 (ONO4). Similar with the total gross rate of organic N mineralization, there were no significant differences in the immobilization of NH+4 into organic N between woodland and orchard soils or orchard soil age. However, ONO4 was stimulated by the conversion of woodland to orchard significantly (p < 0.01, Table 2). Therefore, the net production rate of NH+4 decreased with increasing ONO4 and was negative in most studied soils. Furthermore, a significant exponential relationship was observed between the net NH+4 production rate and NH+4 content, which was determined in fresh soils (p < 0.01, Fig. 6).

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Table 1 – Soil properties of studied orchard and woodland. Soil properties

Woodland

Orchard Orch-2

pH SOC (g/kg) TN (g/kg) C/N NH+4 (mg/kg) NO−3 (mg/kg) Nitrate ratio Nlab (mg N/kg) Nrec (mg N/kg)

5.19 7.18 0.47 15.41 12.38 6.23 0.35 54.13 397.7

± ± ± ± ± ± ± ± ±

0.09 0.99d 0.09c 1.37 5.53a 2.17 0.18c 25.91c 65.3d

4.44 13.40 0.94 14.57 16.06 43.28 0.73 151.7 725.8

± ± ± ± ± ± ± ± ±

0.03 4.53c 0.36b 1.31 8.60a 23.11 0.02b 54.4bc 276.3cd

Orch-10 5.12 14.98 0.97 15.48 2.56 20.52 0.86 121.7 821.5

± ± ± ± ± ± ± ± ±

0.77 2.70c 0.07b 2.32 1.04b 18.73 0.07ab 29.6c 45.2bc

ANOVA analysis Orch-20

4.54 22.06 1.41 15.94 2.61 15.18 0.85 270.7 1123.1

± ± ± ± ± ± ± ± ±

0.64 4.87b 0.40b 2.11 0.22b 2.85 0.03ab 108.4ab 292.3b

Orch-30 4.18 29.84 1.90 15.75 2.11 29.20 0.93 374.8 1491.3

± ± ± ± ± ± ± ± ±

0.07 1.43a 0.11a 0.93 0.47b 4.63 0.01a 75.2a 65.7a

F

P

2.886 20.815 13.870 0.292 6.157 3.257 20.525 11.157 14.953

0.079 0.000 0.000 0.876 0.009 0.059 0.000 0.001 0.000

Data are shown as mean ± S.D.; SOC = soil organic carbon; TN = total nitrogen; Nlab = soil labile organic N; Nrec = soil recalcitrant organic N; nitrate ration was calculated by [NO−3]/([NO−3]+[NH+4]); the different letters in the same line indicate significant difference at p < 0.05 level.

2.4. NO−3 production and consumption Autotrophic nitrification (ONO4) and heterotrophic nitrification (ONrec) were two NO−3 production processes. The 15N tracing study showed that the rates of ONO4 in the orchard soils, ranged from 1.251 ± 0.182 to 2.516 ± 0.886 mg N/(kg·day), were significantly higher than that in the woodland soil (0.129 ± 0.158 mg N/(kg · day)). ONrec was negligible and not different significantly between the woodland soil and orchard soils and between the orchard soils (Table 2). Correlation analysis showed that ONH4 was positively correlated with soil organic C and N contents (p < 0.05 for both), and bacterial and archaeal amoA gene copy numbers (p < 0.01 for both), but not with soil pH (p > 0.05). We defined the ratio of ONH4 to total gross rate of organic N mineralization (MNrec + MNlab) as the nitrification capacity, which indicated the nitrate ratio in inorganic N very well (Fig. 7). The change in nitrification capacity could be best described by the Michaelis–Menten equation with Vmax = 1.676 and Km = 3.071 (Fig. 8). The NO−3 consumption process rates (i.e., INO3 and DNO3) were low with no significant differences between orchard and woodland soils (Table 2). Thus, the differences in net NO−3

1800 Nlab Nrec

Nlab or Nrec (mg/kg)

1500

y = 760.454x + 47.615 r2 = 0.991, p < 0.01

1200 900

y = 232.761x - 69.923 r2 = 0.930, p < 0.01

600 300 0 0.3

0.6

0.9

1.2 1.5 Total N (g/kg)

1.8

2.1

Fig. 2 – Relationships between total N content and labile organic N (Nlab) and recalcitrant organic N (Nrec) in orchard with different ages and adjacent woodland soils.

production rates (NN) between studied soils were mainly dependent on the differences in ONH4. NN ranged from 1.123 ± 0.210 to 2.143 ± 0.215 mg N/(kg·day) in the orchard soils and was significantly higher than −0.070 ± 0.368 mg N/(kg · day) in the woodland (p < 0.01), but the differences between orchard soils were not significant. No significant relationship could be observed between NN and NO−3 content determined in fresh soils (p > 0.05). We defined the net production rate of inorganic N as an index of inorganic N supply rate (NS) and calculated NS by inputting the gross rates presented in Table 2 into Eq. (2). The results showed that the value of NS in majority of the studied soils was negative and the averages ranged from −0.563 mg N/(kg·day) in Orch-30 to −0.104 mg N/(kg·day) in Orch-2. The differences between woodland soil and orchard soils and between orchard soils were not significant (p > 0.05).

3. Discussion 3.1. Inorganic N supply in orchard soils Due to the large amendment rates of organic manure, soil organic C and N increased with increasing orchard age (Table 1). However, the inorganic N supply rate (NS) did not increased with the accumulations of organic C and N, but decreased, on average, with increasing orchard age (Table 2). The change in NS with orchard age was in high agreement with fruit farmers' impression that the demand for N fertilizers did not decrease for citrus plant growth with orchard age. Thus, fruit farmers did not reduce the N fertilizer rates with increasing soil organic C and N contents. Indeed, the application rate was, on average, 400 kg N/ha to the orchards <7 years and increased to 500–600 kg N/ha to the orchard >7 years old. To understand the factors controlling this would facilitate the enhancement of the soil biological inorganic N supply rate and enable N application rates without loss of fruit yield or market quality. N mineralization is the main processes for soil biological inorganic N supply. Accumulated organic N was allocated mainly into Nrec and secondly into Nlab (Fig. 2). As expected, the gross rate of labile organic N mineralization increased with its content increasing in soils, however, the gross rate of

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10

log(amoA copies/g soil)

AOA

A

AOB

8

a

A a

A a

a

B

C

b

6

4

2

0

0

2

10 20 Orchard planting age (year)

30

Fig. 3 – Archaeal (AOA) and bacterial (AOB) amoA gene copy numbers in orchard soil of different age. Values with same letter in the amoA gene are not significant different (p < 0.05). recalcitrant N mineralization did not increase, but decreased with its content increasing in soils (Fig. 4), as a consequence, total gross rate of organic N mineralization remained unchanged with increasing orchard age. Therefore, the negative relationship between Nrec and MNrec was the crucial reason for NS not increasing with increasing soil organic N contents as orchard age increased. Soil organic N mineralization is controlled by the soil environment, such as soil microbial biomass and activity, pH, and C/N ratio (Zech et al., 1997; Arslan et al., 2010). Conversion of woodland to orchard and increasing orchard age, soil pH tended to decrease due to N fertilizers application (Table 1), which is consistent with previous reports (e.g. Guo et al., 2010b). Therefore, high contents of organic N was accompanied by low pH in the studied soils (r = −0.636, p < 0.05 for Nrec and r = −0.707, P < 0.01 for Nlab). Taking the turnover time (TT) as equaling to decomposability of organic N (Eq. (3)), the larger the turnover time, the more stable the organic N pool is. Statistical analysis showed that the turnover time of recalcitrant (TTNrec) decreased exponentially with increasing soil pH significantly, but the

turnover time of labile organic N (TTNlab) was not correlated with soil pH significantly (Fig. 5). Low pH increases the content of Fe and Al oxides in humid subtropical soils (Qafoku et al., 2004), which may be complexed with organic matter through carboxyl group, aliphatic and O-alkyl constituents (Oades et al., 1989; Baldock et al., 1992; Eusterhues et al., 2005). The bonds of Fe and Al oxides and organic constituents are very stable (Boudot et al., 1986). Therefore, low pH may suppress the mineralization of organic N (Fu et al., 1987; Zech et al., 1997). It is already documented that the increase in soil pH by liming can increase N mineralization (Nyborg and Hoyt, 1978). Our results further demonstrated that the suppression effect of low soil pH on mineralization of organic N seemed to be effective only for recalcitrant organic N and ineffective for labile organic N, which was in line with the previous report that there was little influence of pH on arginine (Lin and Brookes, 1999), which was used as a surrogate for labile organic N (Aciego Pietri and Brookes, 2008). Less functional groups such as carboxyl, aliphatic and O-alkyl constituents in light organic matter fractions (Sohi et al., 2001; Poirier et al., 2005), which are defined as labile organic N in the current study, would lead to the ineffectiveness of soil pH on labile organic N mineralization. It has been well known that soil pH affects soil microbial community and their activities as well. However, it needs further investigation as to whether there are differences in the effects of soil pH on mineralization of recalcitrant organic N and labile organic N via affecting soil microbial community and their activities. Therefore, to raise soil pH by liming is an option to increase the gross rates of recalcitrant organic mineralization, thus an increased soil biological inorganic N supply rate and reduce the dependence on chemical N fertilizer application to the orchards. However, raising soil pH increases the rates of nitrification, which increases the risk of N losses through NO−3 leaching and runoff in humid subtropical region. So, the impacts of liming on availability of soil organic N for plants and NO−3 leaching and runoff shall need to be evaluated comprehensively. Additionally, environmental variables and biological properties were important factors effecting organic N mineralization. However, the environmental variables could not be obtained for

Table 2 – Gross and net N transformation rates in studied orchard soils and adjacent woodland soil estimated by 15N tracing model (mg N/(kg·day)). Processes

Woodland

Orchard Orch-2

MNrec MNlab INH4_Nrec INH4_Nlab ONrec ONH4 INO3 DNO3 NS NN

1.071 0.292 0.376 1.150 0.002 0.129 0.201 0.001 −0.363 −0.070

± ± ± ± ± ± ± ± ± ±

0.341 0.151b 0.243 0.697 0.001 0.158d 0.282 0.000 0.370 0.368b

0.834 1.318 1.241 0.889 0.008 1.251 0.135 0.001 −0.104 1.123

± ± ± ± ± ± ± ± ± ±

0.522 0.415a 0.153 0.568 0.003 0.182c 0.096 0.000 0.319 0.210a

Orch-10 1.076 0.726 0.848 0.826 0.007 2.516 0.389 0.001 −0.255 2.130

± ± ± ± ± ± ± ± ± ±

0.634 0.440ab 0.605 0.679 0.005 0.886a 0.155 0.001 0.681 1.035a

ANOVA analysis Orch-20

0.433 0.661 0.996 0.582 0.008 1.489 0.017 0.008 −0.493 1.473

± ± ± ± ± ± ± ± ± ±

0.088 0.194ab 0.612 0.475 0.006 0.602bc 0.013 0.012 0.993 0.601a

Orch-30 0.275 1.310 1.554 0.397 0.039 2.347 0.236 0.007 −0.563 2.143

± ± ± ± ± ± ± ± ± ±

0.141 0.407a 1.554 0.110 0.040 0.451ab 0.238 0.010 1.133 0.215a

F

P

2.478 5.016 1.356 0.836 2.007 9.813 1.657 0.820 0.171 7.512

0.111 0.018 0.316 0.532 0.169 0.002 0.235 0.541 0.948 0.005

MNlab = mineralization of labile organic-N to NH+4; MNrec = mineralization of recalcitrant organic-N to NH+4; INH4_Nrec = immobilization of NH+4 to recalcitrant organic-N; INH4_Nlab = immobilization of NH+4 to labile organic-N; ONrec = oxidation of recalcitrant organic-N to NO−3; ONH4 = oxidation of NH+4 to NO−3; INO3 = immobilization of NO−3 to recalcitrant organic-N; DNO3 = dissimilatory NO−3 reduction to NH+4; NS = inorganic N supply; NN = net rate of nitrification.

16 2.1

y = 0.0021x + 0.4519 r2 = 0.243, p < 0.05

1.8

Nlab Nrec

1.5 1.2

y = -0.0009x + 1.521 r2 = 0.509, p < 0.01

0.9

lab

rec

MN or MN (mg N/(kg soil.day))

J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 4 (2 0 1 5) 10 – 1 9

0.6 0.3 0.0 0

300

600 900 1200 Nlab or Nrec (mg/kg)

1500

1800

Fig. 4 – Relationships between organic N content and gross rate of mineralization in woodland and orchard soils. analyzed, due to space-for-time substitutions were used in our study. Thus, the effects of environmental variables and biological properties on organic N mineralization could be studied in the future.

3.2. Increased NO−3 production in orchard soils It has been well documented that agricultural use stimulates nitrification activity, particularly in humid subtropical soils (Zhang et al., 2013b; Zhong et al., 2007). Application of N fertilizers is an important factor stimulating nitrification activity in the soils (Zaman et al., 1999; Lu et al., 2012b; Zhang et al., 2012a). It was reported that compared to control soils (no N application), a more than 10-fold of the growth of autotrophic ammonia-oxidizing bacteria (AOB) in the soil was stimulated by mineral N fertilizer (Chu et al., 2008; Shen et al., 2008). Generally, it was considered that AOB abundance decreased with decreasing pH (Nicol et al., 2008). In our investigation, the amoA gene copy numbers of archaeal (AOA) and bacterial (AOB) were higher in orchard soil than that in woodland soil and there was an increasing tendency with increasing orchard age (Fig. 3), although soil pH decreased with orchard age. These results are in line with the results observed by Zhang et al. (2012b) in a long-term experiment in rice-

700

a

8000

Turnover time of Nlab (day)

Turnover time of Nrec (day)

10000

wheat-rotation field. This contradictory phenomenon may be due to the stimulatory effects of N fertilization on AOB abundance higher than the restraining effects of soil pH. Additionally, the increasing contents of SOC and TN in soil also could stimulate AOB abundance (Orwin et al., 2010). In agreement with amoA gene copy numbers of archaeal (AOA) and bacterial (AOB) increased, the rates of ONH4 in orchard soils were much higher than that in woodland soils, consistent with the findings of Zhang et al. (2013a) in agricultural soils and Xue et al. (2009) in tea plantation soils. Since the total gross rate of organic N mineralization was kept almost unchanged, the nitrification capacity increased with increasing orchard age (Fig. 8). But, the rates of INO3 and DNO3 did not change significantly (Table 2). The coupling of the increase in nitrification capacity and low NO−3 consumption resulted in a shift in NH+4 dominance in woodland soil to NO−3 dominance in orchard soils (Table 1), which was consistent with the previous observation (Xue et al., 2006; Zhang et al., 2013a). Considering the fact that large amounts of N fertilizers were applied to orchard soils every year and the large annual precipitation, the risk of N losses through NO−3 leaching and runoff were increased in this humid orchard soils. Taking the change in nitrification capacity with time as an index of the stimulatory effects of agricultural use on nitrification, Fig. 8 shows that the stimulation followed the Michaelis–Menten equation, i.e. it was much sharper in the first 10 years (reached 77% of the maximum) after converting woodland to orchard and gradually weakened with time. But even after 30 years, there would still be stimulatory effects on nitrification capacity in the studied orchard. This result confirmed by the change in regularity of amoA gene copy numbers of archaeal (AOA) and bacterial (AOB) in the soil, which was rapidly stimulated in the first 10 years (Fig. 3). Since N fertilizers not only stimulate nitrification activity, but also lead to soil pH decreases (Ju et al., 2007; Guo et al., 2010b), which enhances Al toxicity and increases the production of antimicrobial substances, inhibiting bacterial growth, retained the process of autotrophic nitrification (Rousk et al., 2010). The negative impacts of soil acidification on nitrification were able to counter-balance the stimulatory effects of N fertilization on the nitrification rate. The feedback effect of soil pH change on nitrification could be the reason that stimulatory effects were weakened with increasing orchard age.

6000 -1.343x

y = 742394e

r2 = 0.445, p < 0.01

4000 2000 0 3.9

4.2

4.5

4.8 pH

5.1

5.4

5.7

600

b

y = 1315.6e-0.385x r2 = 0.108, p > 0.05

500 400 300 200 100 0

3.9

4.2

4.5

4.8 pH

5.1

5.4

Fig. 5 – Effects of soil pH on decomposability of recalcitrant organic N (Nrec) (a) and labile organic N (Nlab) (b).

5.7

17

30

2.4

25

2.0

20

Capacity of nitrification

NH4+ content (mg/kg)

J O U RN A L OF E N V I RO N ME N TA L SC IE N CE S 3 4 (2 0 1 5) 1 0– 1 9

y = 12.337e0.5724x r2 = 0.494, p<0.01

15 10 5

1.6 1.2 0.8 y = 1.676x/(3.071+x) r2 = 0.792, p < 0.01

0.4

0 1

0 -1 -2 -3 Net rate of NH4+-N production (mg N/(kg soil.day))

-4

Fig. 6 – Relationship between the net NH+4 production rate and NH+4 content determined in fresh soils.

For reducing the risk of NO−3 leaching and runoff in the orchard, particularly in the oldest orchards in the region, the application of nitrification-inhibitor and controlled-release or slow-release fertilizers should be examined. Additionally, N fertilizers rate would be reduced if biological inorganic N supply rate was increased and reduce the dependence on chemical N fertilizer application to the orchards.

4. Conclusion The gross rates of N transformation occurring simultaneously in citrus orchard soils with ages of 2, 10, 20, and 30 years, respectively, and adjacent woodland soil in the humid subtropical region of China were determined using a 15N tracing study. The results highlighted the fact that inorganic N supply rate remained unchanged after converting woodland into citrus orchards and did not change with orchard age in a subtropical region of Eastern China. However, the SOC and TN contents increased significantly, mainly due to the concomitancy of soil

Ratio of NO3--N to inorganic-N

1.0

0.0 0

5

10 15 20 25 Orchard planting age (year)

30

Fig. 8 – Dynamics of nitrification capacity after converting woodland to orchard and with orchard age.

acidification, which restricted the mineralization of recalcitrant organic N. Thus, the amount of N needed form fertilizers did not reduce with the accumulation of soil organic N in the orchard soil. The gross rate of autotrophic nitrification and nitrification capacity increased significantly after woodland converted to orchard. As a consequence, NH+4 dominance in inorganic N in woodland soil shifted to NO−3 dominance in orchard soils. Under the subtropical climate characterized by high temperate and rainfall, the risk of NO−3 leaching and runoff is expected to increase. Therefore, management practices which enable increases in the availability of organic N for citrus plants are needed to reduce the reliance on N fertilizers.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 41401339, 41330744), the Natural Science Foundation of Jiangsu Province (No. BK20140062) and Fujian Province (No. 2014J01145), outstanding innovation team in Colleges and Universities in Jiangsu Province and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

0.8

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0.6

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