Ecotoxicology (2014) 23:2069–2080 DOI 10.1007/s10646-014-1329-0

Long-term effects of fertilizer on soil enzymatic activity of wheat field soil in Loess Plateau, China Weigang Hu • Zhifang Jiao • Fasi Wu • Yongjun Liu • Maoxing Dong • Xiaojun Ma Tinglu Fan • Lizhe An • Huyuan Feng

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Accepted: 9 August 2014 / Published online: 19 August 2014 Ó Springer Science+Business Media New York 2014

Abstract The effects of long-term (29 years) fertilization on local agro-ecosystems in the Loess Plateau of northwest China, containing a single or combinations of inorganic (Nitrogen, N; Phosphate, P) and organic (Mature, M Straw, S) fertilizer, including N, NP, SNP, M, MNP, and a control. The soil enzymes, including dehydrogenase, urease, alkaline phosphatase, invertase and glomalin, were investigated in three physiological stages (Jointing, Dough, and Maturity) of wheat growth at three depths of the soil profile (0–15, 16–30, 31–45 cm). We found that the application of farmyard manure and straw produced the highest values of soil enzymatic activity, especially a balanced applied treatment of MNP. Enzymatic activity was lowest in the control. Values were generally highest at dough, followed by the jointing and maturity stages, and declined with soil profile depth. The activities of the enzymes investigated here are significantly correlated with each other and are correlated with soil nutrients, in particular with soil organic carbon. Our results suggest that a balanced application of fertilizer nutrients and organic manure (especially those

Weigang Hu and Zhifang Jiao have contributed equally to this work. W. Hu  Z. Jiao  Y. Liu  M. Dong  X. Ma  L. An  H. Feng (&) MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China e-mail: [email protected] F. Wu National Research Center for Conservation of Ancient Wall Paintings and Earthen Architecture Sites, Dunhuang 736200, Gansu, China T. Fan The Academy of Gansu Agriculture, Lanzhou 730070, China

containing P) has positive effects on multiple soil chemical parameters, which in turn enhances enzyme activity. We emphasize the role of organic manure in maintaining soil organic matter and promoting biological activity, as its application can result in a substantial increase in agricultural production and can be sustainable for many years. Keywords Long-term fertilizer application  Soil enzymes  Soil organic carbon  Wheat yield  Loess Plateau

Introduction The Loess Plateau of northwestern China which is an arid region encompasses 1.3 million ha of periodical wheat and corn agricultural land, and produces about 40 % of the local food needs. Therefore, improving the yield of crops while simultaneously maintaining soil fertility in this area is essential for the local agriculture. The application of organic and inorganic fertilizers is utilized primarily to increase soil nutrient availability to crops, and these fertilizers can further affect the population, composition, and function of soil microorganisms (Marschner et al. 2003). Soil microorganisms and enzymes are the primary mediators of soil biological processes, including organic matter degradation, mineralization, and nutrient recycling. They play a vital role in maintaining soil ecosystem quality and functional diversity (Kandeler et al. 1999; Tarafdar and Marschner 1994). Soil enzymes are derived from soil microorganisms, plant roots, soil animal and plant residues, etc.(Kandeler et al. 1999; Tarafdar and Marschner 1994; Ahemad and Khan 2011). They mediate the transformation of elements in the soil into forms required for plant growth(Burns

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1982) and they control the rate of soil nutrient cycling (Marcote et al. 2001; McLatchey and Reddy 1998). Soil enzymatic activities are the candidate ‘‘sensor’’ of soil management practice since they integrate information from microbial status and soil physico-chemical conditions (Aon and Colaneri 2001). Thus, soil enzymes can act as indicators of soil microbiological characteristics, which are important in detecting changes in soil quality and health due to crop rotation, amendments, tillage, and agricultural management(Eivazi et al. 2003; Tarafdar and Marschner 1994). In the background of soil fertility management, longterm fertilizer experiments (LTFEs) are valuable assets for determining yield trends, evaluating changes in nutrient dynamics and balances, predicting soil carrying capacity, and assessing soil quality and system sustainability (Mandal et al. 2007; Blaise et al. 2005; Marcote et al. 2001; Stark et al. 2008). Previous short-term and long-term experiments examining the effects of fertilizer on soil fertility have collected samples at the beginning or at the end of the cropping sequence (Albiach et al. 2000; Mandal et al. 2007; Marcote et al. 2001; Marinari et al. 2000; Kautz et al. 2004). In contrast, few studies have investigated postfertilization soil enzymatic activity during active crop growth stages, using different organic or chemical fertilizer treatments, or at different depths along the soil profile. In the Loess Plateau of China, several researchers have investigated the effects of long-term fertilization on grain yield, nutrients use, and water availability (Fan et al. 2005; 2008; Huang et al. 2003). However, relatively limited information is available concerning changes in soil biological properties under long-term field condition in the agro-ecosystem. The objective of this study is to ascertain the impact of long-term (30-years) applications of mineral fertilizers and organic manure on soil enzymatic activity at different physiological stages of wheat growth and at different depths (0–15; 16–30; 31–45 cm) along the soil profile. We then examine the relationship between fertilizer and various soil characteristics, with particular attention to the role of precipitation, in this farming region.

Materials and methods Experiment site A long-term field experiment was initiated in April 1979 at the Gapping Agronomy Farm, Pingliang, Gansu, China. The site is located in the central part of the Shizi highland plateau (35°160 N and 107°300 E at 1254 m above mean sea level). The experimental area occurs in the semi-humid arid temperate zone and is characterized by mild summers and cold winters, with mean monthly maximum and

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minimum temperatures of 21.1 and -12.9 °C, respectively, and a mean annual temperature of 8 °C. The mean annual precipitation is 560 mm, of which 60 % is confined to a 4-month period from June to September. May and June are the driest periods for crop growth, and light precipitation is common during December and January. The study area is representative of a typical rain-fed farming region completely dependent on precipitation. The soil is referred to as a heilu soil, which occupies 21.3 % of total arable land in Gansu Province, with 5.3 g C kg-1 of SOC. Experimental design and treatments The experiment began in 1979 in a corn crop on land that had been planted with corn the previous year and had been almost unfertilized except with farmyard manure each year before the start of the experiment. Winter wheat and maize have dominated the crop rotation in the area (18 years of winter wheat, 8 years of maize, and 1 year each soybean and sorghum), with wheat growing in 2007 and 2008. The experiment was performed using a randomized design of six treatments with three replicates. The whole experiment encompassed a 0.44 ha area. Each plot had an area of 16.7 9 13.3 m, with a buffer zone of 1.0 m between each plot. The fertilizers were added annually in the autumn, as follows: (1) (2) (3) (4) (5) (6)

CK (control), no manure or fertilizer N, nitrogen fertilizer annually; NP, nitrogen (N) and phosphorus (P) fertilizers annually SNP, straw (S) plus N added annually and P fertilizer added every second year; M, farmyard manure added annually; MNP, farmyard manure plus N and P fertilizers added annually.

Urea was used as the source for N and single superphosphate for P; these were applied at 90 kg N ha-1 and of 30 kg P ha-1 for wheat and corn. Manure was added at 75 t ha-1 (wet weight). Generally, the manure was a mixture of approx 1:5 ratio of wet cattle manure to dry loess soils, so its nutrient content was variable from year to year. The SOC, N, P, and K contents of the manure mixture taken in 1979 were approx 11.37, 1.07, 0.69, and 12.3 g kg, respectively, in dry weight. Although the specific amounts of nutrients added with manure each year were not determined, an application of approximately 75t ha-1 supplied roughly 40 kg N ha-1, 26 kg P ha-1, and 460 kg K ha-1 in manure annually to crops. For the SNP treatment, 3.75 t ha-1 of wheat straw were returned to the soil prior to plowing, and P fertilizer was added with the straw every second year. There was very little wheat straw or corn residue on the other treatments because all crops

Long-term effects of fertilizer on soil enzymatic activity

were harvested at the ground level and removed from the plots before thrashing the grain. The SNP treatment was the only one that had residue returned to the plots. The C content of the straw was 45 % (Bremer et al. 1995), so there was 1.69 t C ha-1 added each year to the SNP treatment (Fan et al. 2008). Soil samples and soil handling The soil samples were taken from each plot at three different physiological stages of the wheat crop, namely, jointing, dough, and maturity, on 26 April, 28 May, and 22 June 2008, respectively. We randomly selected three points in each repeat plot (three replicates per treatment), removed the surface cover, and dug up a 45 cm soil profile. We collected soil at 0–15, 16–30 and 31–45 cm in depths, mixed the soil samples of each point and placed them in plastic bags to be transported to the laboratory. The soil samples were then air-dried, sieved (1 mm) and stored at 4 °C for soil enzymatic activity analysis. All chemical results are the means of triplicate analyses performed after the soil was oven-dried. Soil moisture was determined after drying at 105 °C for 24 h.

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colorimetric determination of the disodium phenyl phosphate (phenol) released when 5 g soil is incubated with 5 ml 0.075 mM disodium phenyl phosphate and 10 ml boric acid buffer (pH 9.6) at 37 °C for 3 h. Absorbance was measured at 578 nm and activity was expressed as lg phenol released g-1 soil h-1. Invertase activity was measured as described by Guan (Guan et al. 1986). 1 g of soil was incubated at 37 °C for 24 h, with 5 ml of phosphate buffer at pH 5.5 and 15 ml of 8 % sucrose. Activity was determined by quantification of the release of glucose with a spectrophotometer at 508 nm. Glomalin was extracted from the soil following the prevailing EEG (easily extracted glomalin) protocol of autoclaving (121 °C) 1 g of soil in 8 ml of 20 mM sodium citrate at pH 7.0 in 10 ml centrifuge tubes for 30 min. Immediately after autoclaving, we centrifuged the tubes at 10,000 g for 15 min, then poured off the supernatant and stored it at 4 °C until analysis. Protein in the supernatant fluid was determined by the Bradford dye binding assay using bovine serum albumin as the standard (Wright and Upadhyaya 1998). Statistical analysis

Chemical analyses Soil pH was analyzed in a 1:5 soil: KCl solution (1 M), soil available P was analyzed by the Olsen method and total P was analyzed following the HClO4-H2SO4 Mo-Sb Colorimetry method. Soil organic C and total N were measured using the CHNS-analyser system (Elementar Vario EL, Elementar Analysensysteme GmbH, Hanau, Germany) with the combustion method at 450 and 1250 °C, respectively (Analysis and Test Center of Lanzhou University).

All dates were analyzed by SPSS 13.0. A three-factor analysis of variance (ANOVA) was used for the statistical analysis of the effects of treatments, growing stages and profile depth on soil enzyme activities. A one-way ANOVA was also used for analysis of enzyme activities, chemical character, and grain yield of wheat. Least significant difference (LSD at P = 0.05) was used to determine whether means differed significantly. Pearson correlation coefficients were calculated between chemical properties and soil enzymes activities.

Soil enzyme analysis Soil dehydrogenase activity was estimated by reducing 2, 3, 5-triphenyltetrazolium chloride (TTC). Five grams of soil sample were mixed with 15 ml of 0.5 % (w/v) 2,3,5TTC and 5 ml Tris-buffer (pH 7.6), and then incubated at 37 °C in darkness for 24 h. Dehydrogenase enzyme converts TTC to 2,3,5-triphenylformazan (TPF). The TPF formed was extracted with 100 ml methanol, the extracts were filtered, and absorption was measured at 485 nm with a spectrophotometer(Ross 1971). Urease activity was measured following the method (Hoffmann 1968). Five grams of soil were incubated with 10 ml of citrate phosphate buffer (pH 6.7) and 5 ml of 10 % urea solution at 38 °C for 3 h. Activity was determined by measuring the released NH4? with a spectrophotometer at 578 nm. Alkali phosphatase activity was assayed using the method described by Hoffmann (1968), which involves

Results Soil chemical characters Manure and balanced mineral fertilization significantly increased the amount of soil total P, but straw and nitrogen addition to the soil alone did not cause an evident increase (Table 1). Fertilization increased the mean values of available P, except for the N treatment. Total N varied little across treatments and the highest value was found in treatments that received organic manure along with the recommended fertilizer. Soil organic C followed a similar trend and was highest in manure amended treatments. The pH values ranged from 7.25 to 7.49, with that of the unfertilized control soil around the lowest at pH 7.25. Manure and fertilizer application increased soil pH significantly, and the values were less in the different

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Table 1 Soil chemical characters affected by manure and fertilizer during wheat development stages at different depths Treatments

Soil chemical character (mg kg-1 soil)a Total P

Available P

Total N

Soil organic C

pH

Soil moisture

CK

515.86 ± 12.75 d

2.03 ± 0.31 c

511.75 ± 35.05 bc

6765.35 ± 127.56 c

7.25 ± 0.01 e

8.89 ± 0.45 ab

N

522.49 ± 14.64 cd

1.45 ± 0.14 c

476.65 ± 14.79 c

6617.83 ± 175.54 c

7.35 ± 0.02 cd

9.64 ± 0.48 ab

NP

632.01 ± 16.08 a

9.81 ± 1.98 b

506.96 ± 12.76 c

7259.74 ± 128.54 b

7.32 ± 0.02 d

8.73 ± 0.47 ab

SNP

537.27 ± 17.04 bd

6.90 ± 1.18 b

468.47 ± 19.98 c

7084.89 ± 153.90 bc

7.36 ± 0.02 c

M

566.69 ± 0.72 bc

7.39 ± 1.43 b

577.25 ± 24.12 ab

7754.63 ± 159.76 a

7.42 ± 0.02 b

10.18 ± 0.68 a

8.80 ± 0.49 ab

MNP

569.72 ± 23.41 b

16.46 ± 2.93 a

587.06 ± 28.28 a

7903.49 ± 269.81 a

7.49 ± 0.02 a

8.67 ± 0.55 b

Stages Jointing

507.27 ± 13.21 b

8.24 ± 1.58 a

510.76 ± 14.99 b

7078.43 ± 164.15 b

7.31 ± 0.01 c

10.73 ± 0.16 b

Dough

570.01 ± 12.81 ab

8.54 ± 1.43 a

585.88 ± 20.23 a

7711.28 ± 99.64 a

7.43 ± 0.01 a

5.91 ± 0.10 c

Maturity

594.74 ± 7.45 a

5.24 ± 0.86 a

468.42 ± 13.60 b

6903.25 ± 120.37 b

7.36 ± 0.02 b

11.31 ± 0.25 a

Depth 0–15 cm

600.65 ± 10.19 a

15.44 ± 1.79 a

564.43 ± 15.48 a

7812.59 ± 104.40 a

7.39 ± 0.02 a

0.095 ± 0.40 a

16–30 cm

563.48 ± 12.77 b

4.12 ± 0.46 b

532.23 ± 13.79 a

7442.59 ± 83.37 b

7.36 ± 0.01 a

0.096 ± 0.41 a

31–45 cm

507.89 ± 11.04 c

2.48 ± 0.31 b

461.41 ± 20.30 b

6437.78 ± 145.71 c

7.35 ± 0.02 a

0.087 ± 0.31 a

a

Data are expressed as mean ± standard error for n = 27. In a column, means followed by the same letter are not significantly different (P \ 0.05) by LSD test

fertilized treatments than in manure treated plots. Soil moisture slightly changed between different treatments with long-term fertilizer management. Our study revealed that soil nutrient content and soil pH also differed by growth stage of the wheat. The highest total P was found at maturity, followed by the dough and jointing stages. Available P did not change significantly during crop growth. Total N and organic C were highest in the soil during the dough stage of the wheat crop followed by the jointing and maturity stages. The mean values of soil pH and soil moisture were significantly different under different crop stages, in the following order: dough [ maturity [ jointing and maturity [ jointing [ dough, respectively. All soil properties except soil pH and moisture declined with the soil depth, suggesting that soil depth does affect some soil characteristics. Soil enzymatic activities Soil enzyme activities were strongly influenced by the long-term application of organic and inorganic fertilizer at different stages of wheat growth and at different soil depths, as evidenced by highly significant F-values (P \ 0.0001) for the treatments, stages, depths and their interaction (Table 2). The activities of all enzymes were generally higher in the fertilized than in the unfertilized treatments, and the application of organic fertilizer produced higher activity than addition of inorganic fertilizer treatments, although there were some exceptions to this trend when soil samples were collected at the jointing stage in the 16–30 cm and 31–45 cm layers.

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Table 2 F-values for effects of manure and fertilizer on soil enzyme activities during different wheat development stages and at different depths F-valuesa

DA

Ure

AkP

Inv

Glo

Treatment (T)

2363.47

344.54

1096.32

776.19

849.32

Stage (S)

1024.74

78.57

230.83

224.83

842.29

Depth (D)

55325.61

388.98

10689.71

8085.41

3771.81

T*S

417.51

55.39

409.64

385.71

137.42

T*D

355.35

11.37

57.01

39.27

91.85

S*D

360.07

17.18

293.72

77.51

105.83

T*S*D

147.47

10.95

54.98

49.30

25.28

0.999

0.971

0.997

0.996

0.994

2

R a

All values significant at P \ 0.0001

DA dehydrogenase, Ure urease, AkP alkaline phosphatase, Inv invertase, Glo glomalin

Dehydrogenase activity was highest in treatments that received manure only at the dough stage in the 0–15 cm layer, and lowest in the N treatment at jointing in the 31–45 cm layer. At 16–30 and 31–45 cm depths of the dough and maturity stages soils, the soil dehydrogenase showed higher activities in the treatments of MNP and NP than others. The effects of different treatments on dehydrogenase activity showed a similar trend at each stage in the 16–30 and 31–45 cm depths (Fig. 1). The highest urease activity at each stage and depth occurred in the MNP treatment, with the exception of the jointing stage in the 16–30 and 31–45 cm layers, and the lowest urease value was observed in the control treatment, with the exception of the jointing stage at 31–45 cm and

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Fig. 1 Dehydrogenase (lg TPF g-1 h-1) activity in soil with different treatments during jointing, dough and maturity stages and at different soil depths (0–15; 16–30; 31–45 cm). Bars represent standard deviation, n = 3. For each depth, values with the same letters are not significantly different (P \ 0.05)

the maturity stage at 0–15 cm. Unlike dehydrogenase, urease activity in the N treatment was generally higher than in the NP and control treatments. The application of manure had the maximum impact in enhancing urease activity in the soil (Fig. 2). The interactions effects of between treatments and stages as well as between stages and depths on alkaline phosphatase activity were higher than main effect of stages of wheat growth (Table 2). Alkaline phosphatase activity showed a trend similar to that of urease; the control treatment soil showed the least activity and the MNP treatment showed the highest. The higher values were found in the MNP, SNP and NP treatments that received balanced-fertilization at the dough and maturity stages of the wheat crop in the 16–31 and 31–45 cm depths (Fig. 3). Invertase activity showed a significant interaction effect between treatments and stages that was higher than the main effect of stages (Table 2). Invertase activity did not vary much across treatments and the highest value was found in the SNP treatments at the maturity stage in the 0-15 cm depth. The lowest value was observed in the control (CK) at the maturity stage in the 31–45 cm depth (Fig. 4). Glomalin content decreased in the following order: MNP [ SNP [ M [ NP [ N at each stage and depth, although differences were not always significant. The EEG

content ranged from 0.07 to 0.74 mg-1, with a mean of 0.38 mg-1. Like dehydrogenase and alkaline phosphatase, the effects of different treatments on glomalin showed a similar trend at each stage at the 16–30 and 31–45 cm depths (Fig. 5). Enzyme activity was highest at the dough stage of the wheat crop followed by the jointing and maturity stages, although differences were only significant for urease and glomalin. We found highly significant differences in soil enzyme activity at different depths for all enzymes; enzyme activity declined with soil depth (P \ 0.001). The average activities of dehydrogenase, urease, alkaline phosphatase, invertase, and glomalin at different depths varied from 11.85 to 50.86 lg TPF g-1 h-1, 112.44 to 184.21 lg NH4?-N g-1 h-1, 13.6 to 24.85 lg Phenol g-1 h-1, 28.23 to 53.45 lg Glucose g-1 h-1, and 0.27 to 0.51 mg BSA g-1, respectively. A correlation matrix (Table 3) shows the existence of a significant relationship (P = 0.01) between soil enzyme activities and soil characteristics. There was a close positive correlation between dehydrogenase and available P. Soil organic C was correlated with all enzymes measured. Urease had a strong positive relationship with pH value. Close relationships were found between dehydrogenase, alkaline phosphatase, invertase, and glomalin.

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Fig. 2 Urease activity (lg NH4?-N g-1 h-1) in soil with different treatments during jointing, dough and maturity stages and at different soil depths (0–15; 16–30; 31–45 cm). Bars represent standard deviation, n = 3. For each depth, values with the same letters are not significantly different (P \ 0.05)

Wheat yield Fertilizer application, both organic and inorganic, significantly increased the amount of wheat yield, and average values for wheat yield ranged from 1733.38 kg ha-1 for the control to 5623.34 kg ha-1 for MNP (Fig. 6). The balanced application of nutrients led to a more pronounced effect on the size of the wheat yield.

Discussion Chemical properties Phosphorus (P) is an essential nutrient in crop production. In our work, total P and available P increased with fertilization, except for the P-deficient treatment. Similar results have previously been found in several other studies (Chu et al. 2007; Whalen and Chang 2002). The reason is that P in fertilizer or other sources tends to be fixed soon after application and becomes mostly unavailable, resulting in low recovery by crops but considerable P accumulation in soils (Alam and Ladha 2004). The highest soil organic C and total N recorded in the MNP treatment, in contrast with previous researches in which soil organic C and total N increased with

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application of manure showed a similar trend with long-term fertilizer experiment (Manna et al. 2005; Martens 1992). In our experiment, the aboveground biomass was harvested and no residues were incorporated in the soil, except in the SNP treatment. The straw residues decomposed rapidly, leading to a lower soil organic carbon than in the manure application treatment (Grignani et al. 2007). The increase in soil organic carbon with the application of manure and fertilizer was a result of greater input of root biomass due to increased crop productivity(Izaurralde et al. 2000). The decrease in soil nutrients down the soil profile is in accordance with our knowledge of agricultural soil. Similar results have also been reported by several other researchers (Agnelli et al. 2004; Ros et al. 2006). Soil pH was significantly influenced by long-term application of manure or mineral fertilizer. The lowest pH value was found in the control, and mineral treated plots had significantly lower pH than the manure plots. The result showed that long-term fertilizer application, especially of N, had acidifying effects causing a decrease in pH. This confirms earlier findings that most N-containing fertilizers tend to acidify soil (Belay et al. 2002), mainly due to the fact that most fertilizers supply N in the form of NH4?, which upon oxidation releases H? ions (Magdoff et al. 1997). Soil moisture showed significant differences under the different wheat development stages in the following

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Fig. 3 Alkaline phosphatase activity (lg Phenol g-1 h-1) in soil with different treatments during jointing, dough and maturity stages and at different soil depths (0–15; 16–30; 31–45 cm). Bars represent standard deviation, n = 3. For each depth, values with the same letters are not significantly different (P \ 0.05)

Fig. 4 Invertase activity (lg Glucose g-1 h-1) in soil with different treatments during jointing, dough and maturity stages and at different soil depths (0–15; 16–30; 31–45 cm). Bars represent standard deviation, n = 3. For each depth, values with the same letters are not significantly different (P \ 0.05)

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Fig. 5 Glomalin (mg BSA g-1) in soil with different treatments during jointing, dough and maturity stages and at different soil depths (0–15; 16–30; 31–45 cm). Bars represent standard deviation, n = 3. For each depth, values with the same letters are not significantly different (P \ 0.05)

Table 3 Linear correlation coefficient between nutrient and soil enzyme activities Variables DA Ure AkP Inv Glo

TP

AP **

0.505

**

0.393

**

0.554

**

0.536 0.461**

TN **

0.749

**

0.503

**

0.609

**

0.569 0.699**

OC **

0.453

**

0.502

**

0.448

**

0.410 0.520**

pH **

0.725

**

0.730

**

0.793

**

0.742 0.840**

DA

Ure

AkP

**

1.00

**

0.641**

1.00

**

0.893**

0.795**

1.00

**

**

0.610** 0.727**

0.883** 0.795**

0.313 0.719 0.500

0.352 0.471**

0.836 0.867**

Inv

Glo

1.00 0.874**

1.00

TP total P, AP available P, TN total N, OC organic C, DA dehydrogenase, Ure urease, AkP alkaline phosphatase, Inv invertase, Glo glomalin ** P \ 0.01

order: maturity [ jointing [ dough. This is mainly attributed to lower precipitation and increased plant water use efficiency. Soil enzymes Soil dehydrogenase activity is only present in viable cells, and it is thought to reflect the total range of oxidative activity of soil microflora and consequently may be an important indicator of microbial activity (Nannipieri et al. 1990). In our study, the highest dehydrogenase activity was measured in treatments that received organic manure along with the recommended fertilizer. This can be attributed to

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the application of a balanced amount of fertilizer nutrients and manure, which improved the organic matter status of soils and in turn enhanced enzyme activity (Manna et al. 2005). Dehydrogenase activity was dependable on soil organic carbon and was significantly correlated with it. Dehydrogenase activities in the N treatments were significantly lower than in the NP treatments. This indicates that inorganic N fertilizer has weaker effects on dehydrogenase activity than P fertilizer or organic manure. Kautz et al. also indicated that dehydrogenase activity was weakly influenced by mineral nitrogen fertilization, possibly due to lower organic matter content and a lack of P(Kautz et al. 2004).

Long-term effects of fertilizer on soil enzymatic activity

Fig. 6 Wheat yield variation after the long-term fertilizer experiment. Bars represent standard deviation, n = 3. Values with the same letter are not significantly different (P \ 0.05)

Urease releases N-NH4? through urea hydrolysis and are essential in the chain of hydrolysis of amino compounds. These compounds are supplied to the soil from plants and to a lesser extent from animals and microorganisms (Sinsabaugh et al. 2008). In our study, the highest mean values for urease activity were found in MNP and M treatments that received manure, followed by the fertilizer treatments of N, SNP and NP that received straw or mineral fertilizer, and the least in the control plots (CK). It is possible that plots amended with mineral fertilizer or manure or both on a long-term basis might have resulted in the higher urease activity (Nayak et al. 2007). Dick et al. also reported an increase in urease with long-term additions of manure and crop residues in a wheat–fallow system(Dick et al. 1988). However, in contrast to our study, the addition of inorganic N caused a decrease in urease activity. Urease catalyzes hydrolysis of urea to produce ammonia that can be used by ammonia-oxidizing bacteria. The ammonia-oxidizers showed higher diversity in the fertilizer treatment soils compared to the control soil (Wu et al. 2011). Phosphatases play a meaningful role in P cycling, because they provide P for plant uptake by releasing PO4 from immobile organic P. The significantly greater activity of alkaline phosphatase in the manure and straw residues treated soil could be attributed to enhanced microbial activity and perhaps diversity of phosphate solubilizing bacteria due to manure input over the years (Mandal et al. 2007). Manure addition to the soil also may have resulted in changes in origin, states, and/or persistence of enzymes in the soil (Parham et al. 2002). In our work, no significant differences were found between chemical fertilizers treated soil and unfertilized soil (CK); this can be explained by the

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inhibition of alkaline phosphatase synthesis by mineral fertilization (Saha et al. 2008). Invertase (saccharase) hydrolyzes sucrose, a common plant disaccharide, into two reducing hexoses (glucose and fructose) with the breakdown of the glycoside bond. Invertase is also involved in C transformation and is linked to soil microbial biomass(Kandeler et al. 1999; Ross 1983). In our experiment long-term fertilization increased invertase activity except in the N treatment. This may be due to lower crop yield production resulting in lower substrate concentrations in the soil. Parthasarathi and Ranganathan also suggested that enhanced invertase activity was due to enhanced microbial activity and high substrate concentrations in soil (Parthasarathi and Ranganathan 2000). Glomalin, a glycoprotein produced by AMF (Wright and Upadhyaya 1998), is a component of the hyphal and spore walls (Driver et al. 2005), and is quantified operationally in soil as glomalin-related soil protein (GRSP) (Driver et al. 2005). It accumulates in soils with a slow turnover soil carbon pool (Rillig et al. 2003; 2001; Wright and Upadhyaya 1998). Manure and straw additions to soil significantly increased soil glomalin, whereas mineral fertilization caused no significant difference relative to the control. A previous study in the same experimental plots showed that the contents of larger sizes of water-stable aggregates increased significantly after applying organic manure, organic manure plus chemical fertilizers, and straws (Huo et al. 2008). Furthermore, glomalin is highly positively correlated with soil aggregate water stability (Wright and Upadhyaya 1998). Thus, organic manure can increase soil glomalin content. Our study revealed that crop growth stages varied significantly with soil enzymatic activities. The highest soil enzyme activity was recorded at the dough stage. This may be attributed to a strong rhizospheric effect on soil enzymes. Soil enzymatic activity declined with depth in all treatments, which may due to the lack of specific substrates and synthesis of the enzymes by root excretions at the deeper layers. Similar outcomes have previously been obtained in agricultural long-term field experiments using sewage sludge compost, different management systems, and also in forest soil profiles (Agnelli et al. 2004; Eivazi et al. 2003). Enzymes are important soil components involved in the dynamics of soil nutrient transformations. Enzyme activities of soils are usually correlated with their soil nutrient contents, especially organic C (Taylor et al. 2002). In our study, soil urease, alkaline phosphatase and glomalin activity were significantly higher with organic manure or combined manure-fertilizer treatments than with monochemical fertilizer or control treatments. The use of manure likely enhances soil organic C and higher levels of organic C stimulate microbial activity, and therefore enzyme

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synthesis. In addition, after 29 years of organic matter application, enzymes could be highly immobilised in the soil. Moreover, the immobilization of enzymes in compost has previously been observed. For example, Garcia et al. found urease and phosphatase enzymes in the organic compost matrix(Garcia et al. 1993). Dehydrogenase activity, which is a good indicator of microbial activity, was closely related to available P. A deficiency of available P limited microbial activity (Zheng et al. 2009). Dehydrogenase activity in the MNP treatment was higher than in the other treatments, which may be due to the higher amount of available P. In our study, there was a high correlation between soil glomalin and OC and TP. Several previous studies have also reported that glomalin in the soil is highly correlated with organic C, and that Olsen P contributes to glomalin as well (Lovelock et al. 2004; Rillig et al. 2001). Given the highly consistent correlation of glomalin with soil C, glomalin can hence be used for detecting C pool changes(Rillig et al. 2003). Soil pH also affects the activity of soil enzymes through its controls on microbial enzymatic production, ionization-induced conformational changes of enzymes, and/or the availability of substrates and enzymatic co-factors (Tarafdar and Marschner 1994). In our study, all the activities of tested soil enzymes were correlated with soil pH. This is consistent with the findings of (Sinsabaugh et al. 2008), who showed that soil pH is the primary control of soil enzyme activity. Crop yield of wheat was significantly influenced by fertilization treatment. All the fertilized treatments significantly improved wheat yields relative to the unfertilized control. The balanced application of fertilizer, manure, or crop residue resulted in a crop yield three fold higher than the control. The yield data clearly demonstrate the superiority of the integrated use of M and mineral fertilizers, which caused the greatest increase in crop production. The beneficial effect of integrated use of NP and M was more pronounced and effective in enhancing the productivity with the advancement of year of cultivation. This is due to the maintenance of the increase in soil nutrient status and soil biological activity, especially soil enzymatic activity. Application of manure along with the chemical fertilizer improved soil organic carbon and also increased all measured soil enzymatic activities. Thus, our results suggest that good soil fertility management can ensure adequate nutrient availability to crops and increase yields.

Conclusions This study shows that soil enzyme activities are affected by the long-term manure and fertilizer treatments, physiological stages of the wheat crop, different soil depths, and the interactions of all three. In general, soil enzymatic

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activities were highest in balanced-fertilization treatments, in particular when manure was applied. The highest enzymatic activities were measured at the dough stage, followed by the jointing and maturity states, although there was no significant difference in some enzymes. All soil enzymatic activities declined with soil depth. Activities of our studied enzymes are correlated with each other and further correlated with soil nutrients, especially soil organic carbon. This implies that the accumulation of organic matter and the recycling of C have substantial effects on the activity of enzymes involved in mineralization of C, N and P. Our results suggest that a balanced application of fertilizer nutrients and organic manure, especially including P, has positive effects on multiple soil chemical parameters, which in turn affected enzyme activity. Straw manure combined with mineral fertilizers, as a replacement for farmyard manure, would help to increase some of physical and biological properties of agricultural soils. We emphasize the role of organic manure in maintaining soil organic matter and promoting biological activity, which results in a substantial increase in agricultural productivity and can be sustainable over many years. Acknowledgments This research was supported by National Basic Research Program (2012CB026105), National Natural Science Foundation (31170482), PhD Programs Foundation of Ministry of Education (2010021111002, 20110211110021), The Fundamental Research Funds for the Central Universities (LZUJBKY-2013-92) in China, and State Key Laboratory of Frozen Soil Engineering, Chinese Academy of Sciences (SKLFSE200901). Conflict of interest of interest.

The authors declare that they have no conflict

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