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Revista de la ciencia del suelo y nutrición vegetal

On-line version ISSN 0718-2791

R.C. Suelo Nutr. Veg. vol.10 no.2 Temuco  2010 

R.C. Suelo Nutr. Veg. 10(2): 150 - 157 (2010)




Zhang, Y.L.1, Chen, L.J.1*, Chen, Z.H.1, Sun, C.X.2, Wu, Z.J.1 and Tang, X.H.3

1Institute of Applied Ecology, Chínese Academy of Sciences (IAE, CAS), 110016 Shenyang, People's Republic of China. *Corresponding author E-mail:
2College of Sciences, Northeastern University, Shenyang 110004, People's Republic of China.
3Agricultural Technology Service Center, Suibin County, Heilongjiang Province, 156200, People's Republic of China.


No tillage is being populanzed for the rainfed maize production in Northeast China. In order to evalúate its effects on the nutrient contents and enzymatic charactenstics in upland soils of Northeast China, surface (0-20 cm) meadow brown soil samples were collected from the plots under no tillage and conventional tillage in a 7-year field experiment under maize cropping in Shenyang, with the soil pH, contents of total C, N, P and S and available N, activities of a- and (β-galactosidase, a- and (β-glucosidase, urease, protease, phosphomonoesterase, phosphodiesterase, and arylsulphatase, and kinetic parameters of (β-glucosidase, protease, phosphomonoesterase, phosphodiesterase, and arylsulphatase determined. Comparing with conventional tillage, no tillage increased the contents of soil total C, N, and S and available N, the activities of test enzymes, and the Vmax/Km of soil urease, protease, and phosphomonoesterase, but decreased the activity of soil a-galactosidase and the VmaxKm of soil (β-glucosidase significantly. All the results suggest that long term no tillage for the maize production on meadow brown soil of Northeast China could enhance soil nutrients storage and the turnover of soil N and P, but had definite negative effects on the transformation of soil C.

Keywords: Conservation tillage, conventional tillage, soil enzyme activity, enzyme kinetic properties.


Northeast China abounds in rainfed maize (Zea mays L) and soybean (Glycine max L.), being one of the most susceptive regions to climate change. Its maize production occupies 28% of China's. The intensive maize cropping based on conventional tillage has been traditional in Northeast China for several decades, which led to the continuous soil degradation with an increasing cost/benefit ratio (Mannering and Fenster, 1983; Stonehouse, 1997; Roldan, et ai, 2005; Wang, et ai, 2006). No tillage, an alternative to conventional tillage, has been adopted by many countries to avoid soil erosión, and offered numerous benefits that conventional tillage could not match (Uri, 2000; Borie et ai, 2002; Wang et al, 2006).

Soil nutrient contents relate to the soil productivity, while soil enzymatic characteristics can provide information about the status of key biochemical reactions that participate in the rate-limiting steps of the transformation of soil nutrients. Soil enzymes had rapid responses to the changes of soil management modes (Angers et al, 1993; Mullen et al, 1998; Marx et al, 2001; Jime'nez et al, 2002; Alvear et al. 2005), being of significance in characterizing the effects of farming system on soil properties (Farrell, et al, 1994).

The aim of present study is to understand the changes in the nutrient contents and enzymatic characteristics of meadow brown soil, a typical agricultural soil for rainfed maize production in Northeast China, under 7-year no tillage and maize cropping, and to evalúate the effects of no tillage on the improvement of soil quality under rainfed farming.



Study site and field experiment

A 7-year field experiment was conducted on a meadow brown soil (Luvisol, FAO, 1982) at National Field Research Station of Shenyang Agroecosystems, Chinese Academy of Sciences (41°31'N, 123°24'E). This Station is located at Lower Liaohe River Plain, with a warm and semi-humid continental monsoon climate. The mean annual air temperature is about 7-8°C, cumulative temperature (>10°C) is 3300-3400°C, mean annual precipitation is 650-700 mm, and non-frostperiod is about 147-164 d.

Two treatments were installed, i.e., conventional tillage (CT) and no tillage (NT). The experimental design was a randomized complete block in a split plot arrangement. Each treatment was in 19.0 m x 9.0 m plots with 4 replicates, and applied with 150 kg N hm2 and 40 kg P hm2. The test crop was maize (Zea mays L).

Soil collection

Surface (0-20 cm) soil samples were collected after maize harvested in October 2005. The fresh samples from at least 15 locations in each plot of each treatment were taken, gently mixed, and sieved to remove root material. Parts of the samples were used for the determination of moisture content, available N (NH4+-N and N03"-N), and enzyme activities; parts of them were air-dried at room temperature and ground for the determination of soil total C, N, and S.

Assay of soil chemical properties

Soil moisture content was determined gravimetrically after drying at 105°C; Soil pH was determined in soil: water suspensión (1: 2.5 ratios) with a glass electrode (Lu, 2000). Soils were analyzed for total carbón (TC), total nitrogen (TN) and total sulfur (TS) with one CNS analyzer - Elementar Vario EL ÜT (Matejovic, 1995). Total phosphorus (TP) was determined by digestión with H2S04-HC104 method (Kuo, 1996). Available nitrogen (inorganic N) (AN) was analyzed using the Continued-Flow Analysis (CFA) after extraction by KC1 (2 mol L-1) (Keeney, 1982).

Assay of soil enzyme activities

Enzyme substrates were purchased from Sigma-Aldrich. Inc, SeeBio Biotech, Inc, and J&K China Chemical Ltd., respectively. Enzyme activities were measured according increase of resultant or decrease of substrate by colorimetric determination methods. Soil enzyme activities were assayed within 2 weeks after sampling. During this time, samples were stored at 4°C. Soil urease (E.C.,phosphomonoesterase (E.C., pH 6.5; PMase) phosphodiesterase (E.C., pH 8.0; PDase), arylsulfatase (E.C., pH5.8; ArSase), α-D-glucosidase (E.C., pH 6.0; α-GLUase), (β-D-glucosidase (E.C., pH 6.0; (β-GLUase), α-D-galactosidase (E.C., pH 6.0; α-GALase) and (β-D-galactosidase (E.C., pH 6.0; (β-GALase) activity was assayed by the method of Tabatabai (1994). Soil samples (6.0 g) were reacted with 0.2% urea as substrate at 37°C for 5 h, and the amount of residual urea was determined by using diacetyl monoxime-antipyrine in KCl-acetic phenyl mercury extract with a continuous flow auto analyzer (BRAN+LUEBBE). For PMase, PDase, ArSase, α-GLUase, (β-GLUase, α-GALase, and (β-GALase, soil sample (1.0 g) was reacted with substrate (50 mM sodium p-nitrophenyl phosphate, sodium Bis-p-nitrophenyl phosphate, potassium -p-nitrophenyl sulfate, p-nitrophenyl α-D-glucoside, p-nitrophenyl, p-D-glucoside, p-nitrophenyl α-D-galactoside, p-nitrophenyl p-D-galactoside as substrates respectively at optimal pH. After incubation for 1 h (37°C), CaCl2-NaOH or CaCl2-Tris (hydroxymethyl aminomethane) was added to stop enzymatic reactions, precipitate humic molecules responsible for brown coloration and extract p-nitrophenol. The colored product was measured colorimetrically at 410 nm. Soil protease (E.C.3.4.21-24, PRase) activity was assayed by the method of Ladd and Butler (1972). Briefly, fresh soil sample (1.0 g) was reacted with 2% Na-caseinate as substrate and Tris buffer for 2 h (50°C) at optimal pH and the residual casein was precipitated with 10 % trichloroacetic acid and fíltrate was reacted with Na2C03 and Folin-Ciocalteu reagent. The tyrosine concentration was measured colorimetrically at 700 nm after lh incubation at room temperature. All the determinations were performed at optimal pH. For all enzyme assays, controls were included for each soil sample analyzed. The same procedure as for the enzyme assay was followed for the controls

Determination of kinetic parameters

Seven concentrations (0.005, 0.010, 0.015, 0.020, 0.035, 0.020 and 0.040 mol-L-1) of urea solution, six concentrations (0.0005, 0.001, 0.0025, 0.005, 0.015 and 0.050 mol-L-1 ) of sodium p-nitrophenyl phosphate solution, six concentrations (0.0005, 0.00075, 0.01, 0.015, 0.03 and 0.050 mol-L-1 ) of sodium bis (p-nitrophenyl) phosphate solution, seven concentrations (0.0005, 0.001, 0.005, 0.01, 0.015, 0.025 and 0.05 mol-L-1 ) of potassium p - nitrophenyl sulfate solution and six concentrations (0.003, 0.007, 0.010, 0.020, 0.030,0.050 mol-L-1 ) of (β-D-Glucoside solution were used as the substrates of urease, phosphomonoesterase, phosphor-diesterase, arylsulfatase and (β-glucosidase, respectively. The kinetic parameters Fmax and Km were calculated by nonlinear regression of the statistical software origin 8.0.

Statistical analysis

All determinations were performed in triplicate, and all the valúes reported are means and expressed by per g oven-dried soil (105°C). Data treatment and statistical analysis were performed by using SPSS 10.0 Program. For each variable measured, the data were analyzed by one-way ANOVA and differences of means were carried out by using the t-test at P= 0.05.



Soil nutrient contents

No significant differences were observed in soil pH, total P and total S between treatments NT and CT, but the contents of soil total C, total N and available N in NT increased significantly compared with those in treatment CT (Table 1).



Soil enzyme activities

Comparing with CT, NT increased the activities of soil urease, protease, phosphomonoesterase, phosphodiesterase and arylsulfatase, but decreased the activity of soil α-D-galactosidase significantly. No significant differences were observed in the soil (β-D-galactosidase, α-D-glucosidase, and (β-D-glucosidase activities between NT and CT (Figure 1).


Soil kinetic parameters

As shown in figure 2, the Wmax of soil protease, phosphomonoesterase, phospho-diesterase and arylsulfatase was higher in NT, while that of β-D-glucosidase and urease was higher in CT. The Km of soil protease, phosphomonoesterase, and phosphodiesterase was higher in NT than in CT, while that of soil urease was in reverse. No significant difference was observed in the Km of soil (β-D-glucosidase, and arylsulfatase between NT and CT (Figure 2).

The NT increased the Vmax/Km of soil urease, protease, and phosphomonoesterase but decreased that of (β-D-glucosidase, and had lesser effects on the Vmm/Km of phosphodiesterase and arylsulphatase (Table 2).





Conventional tillage often destroys soil structure, allowing a faster mineralization of soil organic matter (Alvear et al, 2005), while no tillage can improve soil aggregation (Dao, 1998; Green, et al., 2007). In our study, no tillage increased soil organic matter content, being beneficial to the maintenance of soil structure and the reserve of soil moisture. NT treatment increased five enzymatic activities contributing to the distribution of nutrients and organic C turnover without plowing (Dick et al., 1996). Factors contributing to the higher activities under no-tillage may include the absence of the disturbing, which mitigate the problem of runoff of soil nutrient (Bandick and Dick, 1999). Mullen et al. (1998) pointed that increased total soil organic matter produces an increase in enzymatic activities, especially acid phosphomonoesterase. These results are confirmed by many researchers (Angers et al., 1993; Bandick and Dick, 1999). Higher α-D-galactosidase activity in present study could be the effect of crop residues left on soil, which provide C and substance for these enzymatic activities (Wick et al., 1998), increasing soil humus amount and protecting enzymatic fraction (Martens et al., 1992). Mijangos (2006) reported that higher valúes were found in NT plots except for phosphatase activity. Study of Roldan et al. (2005) showed that NT positively influenced urease and phosphatase, while not influenced dehydrogenase, (β-D-glucosidase and protease.

Our results showed that NT could enhance soil nutrients storage and the turnover of soil N and P, while not C. Similarly with most enzymes activity, Vmax valúes of phosphomonoesterase, phosphodiesterase and arylsulfatase, and Vmax/Km of urease, phosphomonoesterase and phosphodiesterase were higher in NT. Soil urease had lower activity and Vmax, but higher catalytic potential Vmax/Km in NT. Lower kinetic velocity (Vmax) and catalytic potential (Vmm/Km) of (3-D-glucosidase confirmed its' lower activity in NT, although some research (Mullen et al., 1998; Bandick and Dick, 1999; Alvear et al, 2005) showed that (3-D-glucosidase was significantly higher in NT than CT. a-galactosidase, (3-galactosidase, a-D-glucosidase and (3-D-glucosidase play important role in the C cycle in soil, being positively related to total soil carbón in general (Eivazi and Tabatabai, 1990), while our study showed a-galactosidase, (3-galactosidase, a-D-glucosidase had no distinctly relationship with SOC. Seven years' no-tillage for the maize production on meadow brown soil of Northeast China had definite negative effects on the transformation of soil C.

In many soils, increases in various soil enzyme activities have been associated with decreases in tillage intensities, and were highly correlated with C contents (Green et al., 2007). While urease, (3-D-galactosidase, a-D-glucosidase and (β-D-glucosidase activity had no distinctly changes in NT system as compared to CT at the end of 7 years. Therefore, the correlation found in other soils did not hold trae for meadow brown soil.



Our data of chemical, biochemical properties in the surface layer of one soil located at the temperature humid zone (North-China) were coincident and clearly showed a further improvement of soil quality upon no tillage systems over a 7-year period as compared to conventional tillage. Here, it was concluded that soil enzyme characters (activities and kinetic parameters Vmax, Km and Vmm/Km) have great valué as early and sensitive indicators of changes in soil properties induced by different farm management systems. To be practical use, indicators of soil quality must be responsive to soil management practices in a relatively short time. The results also indicate that soil enzyme kinetic properties could be measures as indicators of changes induced by tillage systems.



The authors are grateful for the help received by the anonymous reviewers. This study was supported by the Chinese Government Science and Technology Supporting Program (2006BAD10B01) and NSFC Program (40771004).



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