Preceding crops as a source of organic matter are important sources of micronutrient and can play an important role in the soil fertility and soil cycling of micronutrients. In addition to the role of the organic matter in increasing the concentration of micronutrients in soil solution, attention should be also paid to the role of the kind and the quantity of the root’s exudates released in response to the incorporation of different plant residues in the rhizosphere. Present research was conducted with the objective of studying the effect of the kind of preceding crops: Trifolium (
Trifolium pretense L) and Sorghum (
Sorghum bicolor L) on chemical forms of copper (Cu) in solid phases of a calcareous soil in a completely randomized block field experiment with split plot (3 m × 5 m) arrangement, consisting of 3 replications and 3 treatments. After incorporation of the residue, wheat (genotype back cross) was planted. After harvesting the wheat, soil samples were collected from root zone of wheat. Selected soil properties and chemical forms of Cu were determined in the solid phases of the soil samples. Incorporation of plant residues significantly increased the concentration of DTPA-extractable Cu, in the soil. The highest effect was obtained for Trifolium treatment. Incorporation of plant residues decreased the carbonate-bound Cu (Cu-Carb) fraction in the solid phase and increased oxide-bound Cu (Cu-Ox) as compared to the control (fallow treatment). Fraction of organic-bound Cu (Cu-Org) in the soil increased with incorporation of plant residues as compared with the fallow treatment. Trifolium was the most effective in increasing Cu-Org. Cu-Ox and Cu-Residual (Cu-Res) forms showed a significant negative correlation and Cu-Org showed a significant positive correlation with the concentration of DTPA-extractable Cu. Incorporation of
Trifolium residues decreased the fraction (%) of Cu-Carb and Cu-Ox (less soluble forms) and consequently increased the fraction (%) of Cu-Org which in turn elevated the concentration of DTPA-extractable Cu.
Trifolium was the most effective in increasing the phytoavailability of Cu in soil.
Copper is a vital micronutrient in organisms, particularly plants, most notably because Cu participates in protein synthesis, membrane activities, and photosynthesis [1] [2] . The chemical properties of Cu endow the metal with pivotal roles in cell physiology as a catalytic cofactor in many redox enzymes and as a structural contributor in protein conformation, making Cu an essential micronutrient for plants or even all organisms. Our knowledge of the mechanisms or processes in plants and soils influencing Cu oxidation state, speciation, solubility, mobility, uptake and transport in plants, is still scarce in most cases [3] . Copper is often applied to soil and crops as fertilizer to ameliorate micronutrient deficiencies caused by prolonged, intensive agriculture [4] . Therefore, with regard to the key role of Cu copper in plant, it is necessary that the Cu chemistry be evaluated in soil. There are several mechanisms for bounding Cu in soil solid phase such as carbonates, sulfates, Fe, Al and Mn oxides and organic matter. On the other hand, the distribution of Cu in different chemical forms influences its bioavailability. Cu Copper has also a strong tendency to bind to organic compounds, which in turn can affect its availability for plant uptake. Agbnyn (2010) showed that the characteristic chemical and physical soil organic matter played an important role in formation of Cu forms in the soil [5] . Incorporation of crop residues into the soil as a source of organic matter and micronutrients such as Cu can play an important role in the soil fertility and cycling of micronutrients. Because organic acids released from decomposition of crop residues can alter the mobility and bioavailability of metals such as Cu in the soil by modifying soil pH or by releasing soluble organic compounds to complex Cu. For example, about 50% to 80% of Zn, Cu and Mn taken up by rice and wheat crops can be recycled through residue incorporation [6] . Sequential fractionation can be used to determine the amounts and chemical forms of trace elements, as well as to infer the potential bioavailability and to predict the mobility affecting their movement within the soil profile and into groundwater. The number of studies concerning the effect of crop residues incorporation on the fractions of Cu in the soil solid phase and bioavailablility of Cu for plant are limited. Therefore, the objectives of this study were to investigate the effects of crop residues on: 1) the fractions of Cu in the soil solid-phase. 2) on the Cu bioavailability (DTPA-extractable Cu) in soil.
2. Methods and Materials2.1. Field Experiment
The field selected for experiment was located in an area in the southeast city of Esfahan, in the central part of Iran. This area has a relatively flat topography with medium to heavy textured soils. The climate condition is dry with low annual rainfall (<100 mm) and high evapotranspiration (>1500 mm). The soil in the experimental site was classified as Typic Haplocalcids (Soil Classification Working Group, 1998).
Treatments consisted of control (fallow) plus incorporation of the residues of 2 crops: Trifolium (Trifolium pretense L.) and Sorghum (Sorghum bicolor L.) and subsequent planting of wheat (Back Cross genotype) in 3 replications. The experiment was conducted in a complete randomized block field experiment. This made a total of 9 plots (5 × 3). The air-dried crop residues were crashed into the 0.5 - 2 cm pieces and were completely incorporated into the top soil (0 - 30 cm) of each plot with 7 Mg/ha rate. Wheat was planted 30 days after crop residues incorporation and was harvested the following spring. Crop and soil samples were collected at the time of harvest. Soil samples were air-dried, ground and passed through 2 mm sieve and stored for chemical analysis. Available Cu in the soil samples was extracted by diethylenetetramine-penta-aceticacid (DTPA) and Cu concentration in the extract was determined using atomic absorption spectroscopy [7] . Selected properties of the top soil used in this study are shown in Table 1.
2.2. Sequential Extraction of Soil
Fractionation of soil Cu was carried out using the MSEP method [8] . All the five operationally defined binding fractions (exchangeable, bound to carbonates, bound to Mn-oxides and Fe-oxides, bound to OM and residual) could then be extracted using this method. The details of the MSEP are described in Table 2.
During the extractions, a centrifugation was performed at 3000 RPM to separate extracts from solid. The supernatants were transferred into 50 ml volumetric flask and diluted with DI-H2O, then analyzed with flame atomic absorption spectrometer (AAS) for Cu.
2.3. Data Analyses
All statistical analyses were carried out using SAS program. Comparison of the mean values of treatments were tested using one-way analysis of variance (ANOVA). Significant differences between pairs of mean were identified using the LSD test at 5% level [9] .
3. Results and Discussion3.1. Properties of Plant Residues
Table 3 shows C/N ratio and Cu concentration of crop residues. The highest and lowest carbon to nitrogen ratios were related to Sorghum and Trifolium residues, respectively. The highest concentration of Cu was observed for Trifolium residues. Lower C/N ratio of Trifolium caused rapid decomposition of organic compounds and subsequent production of organic acids resulting in lower pH (Table 3).
3.2. Effect of Plant Residues Incorporation on Soil DTPA Extractable Cu
All the treatments significantly increased the concentration of DTPA-extractable Cu in soil. The highest effect was obtained for Trifolium and then Sorghum treatment (Figure 1).
Incorporation of crop residues decreased soil pH that could increase the solubility and availability of Cu compound. It also increased organic matter content and consequently increased the amount of Cu complexed with organic ligands. In addition, residues contains Cu which could be released upon decomposition of plant residues in soil. Stevenson (1991) reported that application of organic fertilizers increased the concentration and
Selected soil properties
Classification
Clay
Silt
Sand
pH
EC (dS/m)
OM (%)
CaCO3 (%)
N (%)
Olsen-P (mg/kg)
NH4-OACK (mg/kg)
Cu DTPA (mg/kg)
Cu Total (mg/kg)
Typic haplocalcids
42.5
46.3
21.2
7.5
6
0.4
33
0.05
11
289
1.4
35.8
Summary of the Tessier et al. (1979) sequential extraction procedure
Step
Fractions
Reagent
Shaking time and temperature
1
Exchangeable (EXC)
8 ml of 1 M MgCl2 (pH 7) in 1 g soil
2 h at 25˚C
2
Bound to carbonates (CAR)
8 ml of NaOAc (pH 5.0 with HOAc)
5 h at 25˚C
3
Amorphous iron-manganese oxides (OXI)
20 ml of 0.04 M hydroxylamine hydrochloride in 25% acetic acid (pH 2 with HNO3)
6 h at 96˚C
4
Organic-bound (ORG)
5 ml of 30% H2O2 (pH 2), plus 3 ml of 0.02 M HNO3 and 3 ml of 30% H2O2 (pH 2) Cool, add 20 ml of a mixture of 3.2 M NH4Ac and 20% HNO3
2 h at 85˚C
5
Residual (RES)
The residual fraction was digested by 8 ml of a mixture of HCl and HNO3Volume ratio of 3 to 1, therefore they were reached by 0.5 M HNO3
30 min at 25˚C
C/N ratio and Cu concentration of crop residues
Plant residues
C/N
Concentration of Cu (mg/kg)
Soil pH
Sorghum bicolor L.
54.2
4.9
7.3
Trifolium pretense L.
19.2
8.2
7.1
availability of micronutrients in the soil [10] . Eghball et al., (2004) observed that application of organic fertilizers increased the availability of zinc for corn [11] .
3.3. Effect of Plant Residues on Cu Fractions in Soil3.3.1. Exchangeable Cu
Amount of exchangeable Cu in the soil samples was lower than AAS detection limit, therefore could not be measured with sufficient accuracy. Payne et al., (1988) showed that the exchangeable Cu is available for plant. Exchangeable form of Cu is usually the lowest form of Cu in soil [12] . Gankl et al., (2002) and (2003) and Yu and Zhou (2006) have also pointed out this [13] - [15] .
3.3.2. Carbonate-Bound Cu (Cu-Carb)
Crop residues treatments significantly decreased (p < 0.05) Cu-Carb in soil in comparison with the control (Table 4). Largest amount of Cu-Carb was found in Sorghum treated plots while Trifolium treated plots had the lowest amount of Cu-Carb (Figure 2 ). Concentration of Cu-Carb in the Trifolium treated plots significantly decreased 12% in comparison with the control. In this study, Cu-Carb ranged from 0.87 mg/kg in Trifolium treated plots to1.4 mg/kg in the control plot. Ma and Uren (1995) reported that in calcareous soils, the average Cu-Carb is from 0.5 - 1.25 mg/kg of soil [16] . Ramos et al., (1994) reported that average of Cu carb is about 0.92 mg per kg of soil [17] . Reduction of Cu-Carb concentration in soil as the results of crop residue incorporation probably was due to their impact on reducing soil pH, which in turn might have increased the solubility of carbonate compounds in soil. Trifolium-treated plots was more effective than Sorghum-treated plots in reducing soil pH that could be the reason for the higher effect in reducing the concentration of Cu-carb (Table 3). Mehra and Jackson (1960) reported that Cu in calcareous soils appeared to precipitate as carbonate or hydroxides [18] . Wei et al., (2006) reported that available Cu was negatively correlated with soil available P and CaCO3 content and a positively correlated with pH. In this study, Cu-Carb was from 0.87 mg/kg in Trifolium treatment and 1.4 mg/kg in control [19] .
The effect of crop residues on the concentration of DTPA-extractable Cu
Analysis of variance of different fractions of Cu (mg・kg<sup>−</sup><sup>1</sup>) in soil as affected by Incorporation of crop residues treatments
Mean Square
Source of Variation
df
Cu-Carbonate
Cu-Oxide
Cu-Organic
Cu-Residual
Replication (RE)
2
0.018
0.12
0.019
3
Crop residues (P)
2
0.12*
1.17***
5.72***
3.77
Error
6
0.53
0.039
1.094
1.81
***, **, *Sighnificant at 0.001, 0.01 and 0.05 levels, respectively.
3.3.3. Fe and Mn-Oxides
Sorghum treatment significantly increased (p < 0.001) the concentration of soil Cu-Ox in comparison with the control treatment. Trifolium treatment, on the other hand, significantly decreased the concentration of soil Cu-Ox as compared with the control (Figure 3). Indeed the concentration of soil Cu-Oxide by Trifolium treatment decreased 14%, also increased 5.3% by Sorghum treatment in comparison with the control. Reduction of the concentration of soil Cu-Ox by Trifolium treatment may have been due to its effect on increasing the concentration of Cu-Org in soil. Indeed, when Cu is added to the soil, a series of interactions (e.g., adsorption, precipitation, complexation, etc.) would take place. The metal forms, associated with Fe, Al and Mn oxides, or bound with organic matter, could be considered potentially active or strongly bound, depending on the physical properties of soil [20] - [25] . Therefore, the form of Cu-Ox can act as a buffering capacity and potential for Cu in soil.
3.3.4. Organically Bound Cu (Cu-Org)
Concentration of Cu-Org significantly (p < 0.001) increased with the crop residues incorporation as compared with the control (Table 4). The largest amount of Cu-Org was found for the Trifolium treated plots (Figure 4). Indeed in the Trifolium and Sorghum treated plots Cu-Org significantly increased 72% and 50%, respectively in comparison with the control.
The effect of crop residues on Cu-Carbonate
The effect of crop residues on Cu-Oxide
The effect of crop residues on Cu-Organic
Higher increase of Cu-Org in Trifolium-treated plots was probably due to the lower C/N ratio of trifolium and consequent increase in decomposition of the added residues resulting in higher soluble organic matter and higher complexation of Cu by organic matter. Krishnamurti and Naidu (2002) showed that the partition coefficient of soil Cu was strongly influenced by the fraction complexed with soil organic matter [26] .
Kawasaki et al. (2000) reported that most of the Cu in industrial effluents, are in organic form [27] . McGrath et al., (1998) demonstrated that increase in soil organic matter resulted in the transfer of copper from other forms to organic form [28] .
Part of this Cu (Cu bound to fulvic acids) can move to soil solution and exist as a part of dissolved Cu which is assumed to be an important factor in the phytoavailability of soil Cu. Tao et al., (2003) in reported that the results of the five-step sequential extraction process followed the order: Organic (47.4%), Residual (30.8%), Oxide (15.9%), Carbonate (5.4%), and Exchangeable (0.5%) [29] . Other authors have already stated that the Cu-Org is the dominant fraction (mean 40.7%) in soils [30] .
3.3.5.Residual Cu Form (Cu-Res)
Incorporation of Trifolium residues decreased Cu-Res in comparison with the control (Table 4) although the decrease was not significant (Figure 5). In this study around 90% and 66% of Cu fractions were in the form of Cu-Res in control and in Trifolium treatment, respectively.
Copper has a stronger affinity to associate with the crystalline structures of the minerals and the organic ligands. As Fuentes et al., (2004) and Nemati et al., (2009) have also pointed out [31] [32] . Burt et al. (2003) reported that around 93% of Cu and 41% of Zn was recovered as residual fraction and very low percentage of Cu was recovered as water soluble and exchangeable fractions [33] . Schramel et al. (2000) found that the copper bound in the residual phase in an uncontaminated soil was around 65% - 85% [34] . Review effect Trifolium on other forms of copper in soil identified that likely the effect of Trifolium on Cu-Organic with compared to Sorghum cause that decreased Cu-residual.
Overall distribution of Cu among different fractions followed the order below:
Cu-Res > Cu-Ox > Cu-Org > Cu-Carb
3.4. Relationships between Forms of Cu in Soil Solid Phase with DTPA-Extractable Cu
Cu-Carb showed no significant correlation with DTPA-extractable Cu in soil. Cu-Ox and Cu-Res forms showed a significantly negative correlation (0.05 and 0.01), respectively with DTPA-extractable Cu (Table 5). Amount of bioavailable Cu reduced by carbonates and oxides [35] . Cu-Org form showed a significant positive correlation (0.01) with DTPA-extractable Cu. This suggests that crop residues incorporation resulted in an increase in soil organic matter and consequent increase in the amount of Cu complexed with organic matter and bioavailable in
The effect of crop residues on Cu-Residual
Regression coefficient (r<sup>2</sup>) between Cu fractions and the concentration of DTPA-extractable Cu in soil
Cu-Carbonate
Cu-Oxide
Cu-Organic
Cu-Residual
0.56ns
0.63*
0.78 **
0.72**
**Significant at p < 0.01; *Significant at p < 0.05; nsNon significant.
soil. Gunkel et al., (2003) reported that organic forms of Cu are most correlated with available forms of Cu for plant uptake.
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