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Published in Agron J 99:1041-1047 (2007)
DOI: 10.2134/agronj2006.0332
© 2007 American Society of Agronomy
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Waste Management

Effect of Liquid Cattle Manure on Corn Yield, Composition, and Soil Properties

Anastasios S. Lithourgidisa, Theodora Matsib,*, Nikolaos Barbayiannisb and Christos A. Dordasc

a University Farm, Aristotle Univ. of Thessaloniki, Thermi 57001, Greece
b Soil Science Lab., Faculty of Agriculture, Aristotle Univ. of Thessaloniki, Thessaloniki 54124, Greece
c Lab. of Agronomy, Faculty of Agriculture, Aristotle Univ. of Thessaloniki, Thessaloniki 54124, Greece

* Corresponding author (thmatsi{at}agro.auth.gr)

Received for publication November 23, 2006.

    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The effects of liquid dairy cattle (Bos taurus) manure on corn (Zea mays L.) yield and composition were studied in a 4-yr field experiment conducted under a Mediterranean environment. In addition, long-term impact of (8-yr) manure application on soil-available NO3–N, P, and K; organic C; Kjeldahl N; and salinity was investigated. Four treatments were established in plots, previously used for a similar 4-yr experiment with winter wheat (Triticum aestivum L.). Treatments were: (i) application of 80 Mg manure ha–1 yr–1; (ii) single application of the equivalent N–P as inorganic fertilization (260 kg N ha–1 yr–1 and 57 kg P ha–1 yr–1); (iii) identical to (ii), but with split N application; and (iv) no fertilization. Corn grain and silage yields, N–P–K plant concentration, and uptake were significantly increased by manure or inorganic fertilizer addition relative to the control. During the 4-yr corn experiment, the amounts of available NO3–N in the soil profile of manure plots were higher than control, but similar to both inorganic fertilization treatments. Manure application maintained the amounts of soil available NO3–N, P, and K at desirable levels, almost each year of the total 8-yr application. However, soil organic C and Kjeldahl N remained unchanged. At the end of the experiment, soil salinity below 30 cm was significantly increased on manure or inorganic fertilizer addition relative to the control, but at levels acceptable for most crops. In conclusion, soil application of liquid dairy cattle manure at a rate equivalent to the recommended inorganic fertilization can enhance corn yield and composition and maintain soil fertility at desirable levels, without increasing soil salinity at unacceptable levels.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
APPLICATION of cattle manure (solid or liquid) to soil can improve soil chemical and physical properties (Sutton et al., 1986; Randall et al., 2000; Eghball, 2002; Butler and Muir, 2006), and consequently, increase crop yields, plant nutrient concentration, uptake (Evans et al., 1977; Beauchamp, 1983; Griffin et al., 2002; Butler and Muir, 2006).

The beneficial effect of cattle manure on crop yield and growth is similar or lower than inorganic fertilizers and this is dependent on the manure N applied (Beauchamp, 1986; Sutton et al., 1986; Randall et al., 2000; Griffin et al., 2002). In general, crop availability of N in manure is lower than N from inorganic fertilizers, because of the slow release of organically bound N and the volatilization of NH3 from surface-applied manure (Beauchamp 1983; Jokela, 1992). Compensation for this reduced availability is generally achieved through increased manure N application rates, in comparison to the N fertilizers (Evans et al., 1977; Beauchamp, 1986; Sutton et al., 1986; Zebarth et al., 1996). However, similar crop yield and plant uptake of macronutrients were obtained after soil application of liquid cattle manure at rates equivalent to the recommended inorganic fertilization (Beauchamp, 1986; Randall et al., 2000; Matsi et al., 2003).

Although both solid and liquid dairy manures are valuable sources of nutrients for crop production (Sutton et al., 1986), liquid manure was found to be better than solid in promoting plant growth (Beauchamp, 1986; Zhang et al., 2006). This is probably due to the higher quantity of immediately available N in liquid than solid manure (Sutton et al., 1986). Beauchamp (1986) reported that average NH4–N/total N ratios were 0.53 for liquid dairy cattle manure and 0.09 for solid beef cattle manure. To preserve the ammoniacal-N from applied manure, especially in the case of liquid manure, injection or incorporation into the soil immediately after application is recommended (Beauchamp, 1983; Randall et al., 2000).

The beneficial effect of cattle manure (solid or liquid) on soil is connected to the enhancement of soil fertility and the improvement of soil structure through the addition of organic matter (Sutton et al., 1986; Randall et al., 2000; Eghball, 2002; Butler and Muir, 2006). However, long-term heavy application rates of cattle manure can have adverse effects on plants (Chang et al., 1991, 1993) and can increase soil salinity and NO3–N leaching, especially in the case of liquid manure (Evans et al., 1977; Chang and Entz, 1996; Vellidis et al., 1996).

Crop nutrition and soil properties are two important factors that must be taken into consideration, for the proper agronomic use of cattle manure as a fertilizer. In addition, any effect of manure application on crops and soil properties should be evaluated in any particular environment. The objectives of this study were to evaluate the effects of liquid dairy cattle manure on corn grain and silage yields, and macronutrient concentration, and uptake, in comparison to those of commercial fertilizers (both applied at equivalent N recommended rates), by means of a field experiment that was conducted under a Mediterranean environment. Since the field where the corn experiment was established had a manure application history, long-term (8-yr) effects of manure on soil-available NO3–N, P, and K; organic C; Kjeldahl N; and salinity were also studied.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Soil and Manure
Matsi et al. (2003) described the site, manure, and soil used for the field experiment. Briefly, the experimental field was located at the Farm of the Aristotle University of Thessaloniki. The soil was classified as Typic Xerorthent (USDA-NRCS, 1996) and was a calcareous loam, with average pH values (1:2 soil/water ratio) 8.3 ± 0.1; CaCO3 content 71 ± 11 g kg–1; and sand, silt, and clay content 259 ± 6, 476 ± 2, and 265 ± 1 g kg–1, respectively. Mean values of selected properties from the liquid dairy cattle manure were pH 7.8 ± 0.1, dry matter 80 ± 3 g kg–1, organic matter 56 ± 1 g kg–1, Kjeldahl N 3.1 ± 0.1 g kg–1, NH4–N 1.3 ± 0.1 g kg–1, P 0.68 ± 0.02 g kg–1, and K 2.5 ± 0.1 g kg–1. All previously reported characteristics of manure are expressed on a wet-weight basis with the exception of pH.

Field Experiment
A 4-yr field experiment with corn (Pioneer hybrid PR3394) was conducted during the years 2002 through 2005. Corn was sown at a seeding rate of 85 000 seeds ha–1 within the last week of May for each growing season. The distance between rows was 80 cm and the size of the experimental plots was 5.6 by 8.0 m with a 2.0-m buffering zone. The experimental design was randomized blocks with four fertilization treatments replicated six times.

The treatments, established in the same plots each year, were: (i) manure, application of 80 Mg ha–1 yr–1 as liquid dairy cattle manure (wet-wt. basis), before sowing; (ii) N-single + P, application of 260 kg N ha–1 yr–1 and 57 kg P ha–1 yr–1, as a single basal dressing before sowing; (iii) N-split + P, application of 130 kg N ha–1 yr–1 and 57 kg P ha–1 yr–1, as basal dressing before sowing and 130 kg N ha–1 yr–1 as side dressing, at the V8 growth stage of corn (first week of July); and (iv) control, no organic or inorganic fertilization.

The rates of inorganic N and P fertilizer applied are recommended for corn, and inorganic fertilizers used were ammonium sulfo-phosphate (20–10–0), super-phosphate (0–20–0), and ammonium nitrate (33.5–0–0). The rate of manure application was based on its Kjeldahl N content and consequently, the amounts of N, P, and K applied to soil in the manure form were almost 250, 55, and 200 kg ha–1 yr–1, respectively. Inorganic fertilizers and manure were incorporated with a tandem harrow disc to a depth of 12 to 15 cm within 2 h after application. Adequate water was applied to corn by surface irrigation and weed control consisted of atrazine (6-chloro-N2-ethyl-N4-isopropyl-1,3,5-triazine-2,4-diamine) applied at 2.25 kg a.i. ha–1 preemergence and rimsulfuron [1-(4,6-dimethoxypyrimidin-2-yl)-3-(3-ethylsulfonyl-2-pyridylsulfonyl)urea] (Rush 25 WG at 50 g a.i. ha–1) applied post emergence. Climatic conditions during the growing periods for corn are reported in Table 1.


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Table 1. Climatic data during the corn growing season for the 4 yr at Thessaloniki, Greece.

 
The field was uncultivated the year before the establishment of the corn experiment (2001) and tandem harrow disc was used to control weeds during this period. However, the field had been used for a similar fertilization experiment with winter wheat (cv. Yecora) during the years 1997 to 2000 (Matsi et al., 2003). The size and the number of experimental plots were the same. In addition, the treatments were similar to these reported in the present study and had been established in the same respective experimental plots used in this experiment. However, rates of manure (40 Mg ha–1 yr–1) and inorganic fertilizers (120 and 26 kg ha–1 yr–1, N and P respectively, single or split application of N) used in the previous study were different and appropriate for winter wheat.

Plant populations were measured in each plot 4 wk after sowing each year of the experiment. Aboveground biomass was collected from two rows of each plot (a 12.8-m2 area) at the kernel milk line stage (around the middle of September). A 1-kg random sample of the biomass was dried at 65°C until constant weight and silage yield (dry-wt. basis) was calculated. The biomass sample was ground to pass a 0.2-mm sieve and analyzed in duplicate for total N by the Kjeldahl method (Bremner, 1996). In addition, duplicate 0.5-g subsamples were ashed at 500°C for 6 h; the ash was dissolved in 2 M HCl, filtered, and analyzed for P by the molybdenum blue–ascorbic acid method (Olsen and Sommers, 1982), and K by flame emission spectroscopy. Plant uptake of N, P, and K was calculated from the plant concentration of each nutrient and the silage yield. Grain yield was determined by harvesting two middle rows of each plot (a 12.8-m2 area) by hand at the end of October and adjusting grain moisture to 15%.

Each year before sowing and fertilization and the last year of the experiment after the growing period, composite soil samples consisting of three subsamples were collected from each experimental plot and from three depths (0–30, 30–60, 60–90 cm). All soil samples were air-dried and ground to pass a 2-mm sieve. The soil samples that were collected from the three depths each year before sowing were analyzed in duplicate for NO3–N, by extraction with 2 M KCl (Mulvaney, 1996) and determination by the ultraviolet spectrophotometric screening method (Clesceri et al., 1998). In addition, Olsen-P (Olsen and Sommers, 1982) and exchangeable K (Thomas, 1982) were determined, in the surface soil samples collected during the same periods. Electrical conductivity in the saturation extract (ECe) (Rhoades, 1996) was measured in the soil samples that were collected from the three depths in the beginning and at the end of the field experiment, and organic C (Walkley and Black, 1934) and Kjeldahl N (Bremner, 1996) were measured in the surface soil samples that were collected during the same periods.

For each plant or soil parameter determined in each year, analysis of variance (ANOVA) was conducted using the program SPSS (version 12). Bartlett's test was performed to check for homogeneity of variances of each parameter among years and the LSD test was used to detect significant differences among means. For each parameter where variances were not statistically different among years, a common LSD value (for all years) was calculated and used for mean comparisons, whereas for each parameter where variances were statistically different, different LSD values (for each year) were used.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Liquid Cattle Manure Effect on Plant Parameters
The number of plants per hectare, determined 4 wk after sowing, were not affected by manure or inorganic fertilization during any year of the experiment. The overall means were 69549 ± 4606, 70241 ± 5159, 69998 ± 4987 and 69546 ± 6114 plants ha–1, for manure, N-single + P, N-split + P, and control treatments, respectively.

Analysis of variance for corn grain and silage yields revealed significant differences among fertilization treatments the last 2 yr (2004 and 2005) of the experiment. For grain yield, F test was significant at p = 0.03 and 0.006, for the years 2004 and 2005, respectively, and for silage yield, it was significant at p < 0.001 both years. Specifically, corn grain yields in the manure treatment were significantly increased relative to the control during years 2004 and 2005 and were similar to the inorganic fertilizer treatments, single or split N application (Fig. 1 ). The same was evident for corn silage yield (expressed as dry biomass) (Fig. 2 ) and N and P plant uptake at the kernel milk line stage (Table 2). At this growth stage, corn uptake of K in the manure treatment was significantly increased compared with the control or inorganic fertilization treatments all years of the experiment except for 2002 (Table 2).


Figure 1
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Fig. 1. Corn grain yield during the 4 yr. Manure, injection of 80 Mg ha–1 yr–1 (wet-wt. basis) liquid dairy cattle manure before sowing; N-single + P, application of 260 kg N ha–1 yr–1 and 57 kg P ha–1 yr–1, as single basal dressing before sowing; N-split + P, application of 130 kg N ha–1 yr–1 and 57 kg P ha–1 yr–1, as basal dressing before sowing and 130 kg N ha–1 yr–1, as side dressing at the V8 growth stage of corn.

 

Figure 2
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Fig. 2. Corn silage yield (expressed as dry biomass) during the 4 yr. Manure, injection of 80 Mg ha–1 yr–1 (wet-wt. basis) liquid dairy cattle manure before sowing; N-single + P, application of 260 kg N ha–1 yr–1 and 57 kg P ha–1 yr–1, as single basal dressing before sowing; N-split + P, application of 130 kg N ha–1 yr–1 and 57 kg P ha–1 yr–1, as basal dressing before sowing and 130 kg N ha–1 yr–1, as side dressing at the V8 growth stage of corn.

 

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Table 2. Plant uptake of N, P and K by corn, at the kernel milk line stage, during the 4-yr experiment at Thessaloniki, Greece.

 
The lack of differences obtained for the plant parameters among fertilization treatments during years 2002 and 2003 were attributed to residual soil available macronutrients, N in particular. In the beginning of the experiment, the levels of soil available N, as it was judged by soil NO3–N, could be characterized as high (see next section). Figures 1 and 2, and Table 2, indicate a decline of plant parameter values in the control the last 2 yr and a similar decline was also observed for the soil available macronutrients (see next section).

Although the results were not consistent over the 4 yr of the experiment, increased corn yields and plant uptake observed in the manure treatment were attributed to the maintenance of soil available macronutrients. This is in agreement with the findings of other researchers, who used liquid cattle manure as a fertilizer for corn (Culley et al., 1981; Beauchamp, 1986; Sutton et al., 1986; Motavalli et al., 1989; Zebarth et al., 1996; Randall et al., 2000). The reported liquid manure application rates are variable and usually higher than the rate used in the present study. However, increased corn yields were reported on manure application at equivalent rates of total N similar or lower than the 250 kg N ha–1 yr–1 rate used in this study. Specifically, increased corn grain yields similar to inorganic fertilizers were obtained with the addition of liquid cattle manure at an equivalent total N rate of 200 kg ha–1 yr–1 for 3 yr (Beauchamp, 1986), {approx}270 kg ha–1 yr–1 for 5 yr (Sutton et al., 1986), and 154 to 224 kg ha–1 yr–1 for 4 yr (Randall et al., 2000). In addition, Zebarth et al. (1996) reported that an application of 175 kg N ha–1 as liquid cattle manure resulted in corn dry matter yields of no less than 90% of the maximum yield. In agreement with the findings of the present study, Culley et al. (1981) and Motavalli et al. (1989) reported that on addition of liquid cattle manure N, P, and K uptake by corn plants increased at levels similar to the recommended inorganic N–P–K fertilization.

Concentrations of N, P, and K in aboveground corn biomass, at the kernel milk line stage, followed the same pattern as plant uptake of the three macronutrients. Specifically, on addition of manure, N and P concentration in plant biomass was increased in comparison to the control and it was similar to that of both inorganic fertilization treatments during 2004 and 2005. Corn K concentration in manure treatment was significantly increased relative to the control or inorganic fertilization treatments for all years of the experiment except 2002 (Table 3). These results are in agreement with the findings of Sutton et al. (1986), who reported that corn leaf N and P concentrations tended to reflect N and P rates applied with liquid dairy cattle manure and the inorganic fertilizer treatment compared with the check, but not consistently each year of the 6-yr field experiment. Evans et al. (1977) reported results that are more consistent and obtained significantly increased N, P, and K levels in corn tissues from liquid beef manure relative to fertilized or unfertilized treatments, but after heavy manure applications. In most cases, the values of all plant parameters determined were similar between the single and split application of inorganic N fertilizer.


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Table 3. Concentration of N, P, and K in corn, at the kernel milk line stage, during the 4-yr experiment at Thessaloniki, Greece.

 
Liquid Cattle Manure Effect on Soil Properties
As mentioned previously, the same field used in this study for the corn experiment had been used for a similar 4-yr fertilization experiment with winter wheat (Matsi et al., 2003). Since the field remained uncultivated for 1 yr between the two experiments, eight annual applications of the liquid dairy cattle manure (40 Mg ha–1 for 4 yr, 1 yr blank, and then 80 Mg ha–1 for 4 yr) were made to the same plots until the end of the corn experiment. Therefore, a part of the following discussion deals with the long-term effects of (8-yr) manure application on selected soil properties.

Soil-available NO3–N, in the beginning of each corn-growing season, was affected by fertilization treatment, depth, and their interaction (Table 4). With the exception of 2003, the lowest concentrations of NO3–N among depths were measured in the 30- to 60-cm soil depth across all treatments. In the 0- to 30- and 60- to 90-cm depths, NO3–N concentrations in manure plots were significantly higher compared with the control, and similar to both or one of the two inorganic N fertilization treatments. However, in certain cases across all soil depths, NO3–N concentrations in manure plots were similar to the control, particularly in the 30- to 60-cm soil depth. In addition, in the same soil depth the concentrations of NO3–N in manure plots were lower than the inorganic fertilization treatments.


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Table 4. Concentration of soil available NO3–N, in the beginning of each growing season, during the 4-yr experiment at Thessaloniki, Greece.

 
These results indicate that application of liquid cattle manure at the recommended N fertilization rate decreases the risk of groundwater contamination due to NO3–N leaching, compared with equivalent inorganic N fertilizer. This is in agreement with the findings of Beauchamp (1983), Jokela (1992), and Randall et al. (2000), who concluded that liquid cattle manure, when applied at optimal rates (the equivalent total N rates used ranged from 224 to 280 kg ha–1 yr–1), does not lead to greater losses of N in subsurface drainage water compared with inorganic fertilizers. In addition, Safley et al. (1985) suggested manure application rates below 224 kg N ha–1 for maximum manure N efficiency by corn, and Zebarth et al. (1996) concluded that selection of proper spring application rates for liquid manure and inorganic fertilizer were found to be equally important in minimizing soil NO3–N. Moreover, Motavalli et al. (1985) and Comfort et al. (1987) found higher amounts and faster downward movement in the soil profile of inorganic N from commercial fertilizer than from injected dairy manure rates applied at comparable N rates.

Increased concentrations of NO3–N at high levels in the soil profile are expected after heavy applications of liquid cattle manure (Evans et al., 1977; Sutton et al., 1979; Culley et al., 1981; Sutton et al., 1986; Vellidis et al., 1996). However, Phillips et al. (1981) reported no greater NO3–N in tile-drain effluent from silage corn receiving 897 kg N ha–1 as liquid dairy manure than from 134 kg N ha–1 applied as inorganic fertilizer. In addition, Beauchamp (1986) reported that application of liquid cattle manure at a rate of 600 kg total N ha–1 did not generally cause surface soil NO3–N levels to be higher than those for urea or the lower rates of manure.

It is worthy to note that the concentrations of soil-available NO3–N, determined in the beginning of the corn experiment in all treatments across the soil depths 0 to 30 and 60 to 90 cm could be characterized as high (Table 4), probably because of the preceding fallow year. This was considered the main reason for no differences obtained for certain plant parameters the first year of the experiment (see previous section). Table 4 indicates a decline of the residual NO3–N in the control with time and this decline was more pronounced the last 2 yr (2004 and 2005) of the experiment.

Apart from the first year of the corn experiment, Olsen P and exchangeable K, determined in the surface (0–30 cm) soil samples of manure treatment, were significantly increased compared to the control and inorganic fertilization treatments, in the beginning of each growing season (Table 5). Evans et al. (1977), Culley et al. (1981), Sutton et al. (1986), Comfort et al. (1987), and Randall et al. (2000) reported similar results. As far as the two inorganic fertilizer treatments were concerned, Olsen P concentrations were increased relative to the control the last 2 yr (2004 and 2005) of the experiment.


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Table 5. Concentrations of soil available P and K (0- to 30-cm depth), in the beginning of each growing season, during the 4-yr experiment at Thessaloniki, Greece.

 
Concentrations of three soil-available macronutrients determined in the 30 cm of soil from manure plots during the corn experiment ranged at levels similar to those of the preceding wheat experiment (Tables 4 and 5). The ranges of the respective concentrations over the 4-yr wheat experiment were 16 to 25 mg kg–1 NO3–N, 13 to 26 mg kg–1 Olsen P, and 76 to 113 mg kg–1 exchangeable K (Matsi et al., 2003). Therefore, it was concluded that no clear trend of nutrient buildup was evident after eight annual applications of the liquid dairy cattle manure. However, as is evident from the data of the present study for corn and as reported by Matsi et al. (2003) for wheat, manure application maintained the amounts of soil-available NO3–N, P, and K at levels similar or higher than the recommended inorganic fertilization for the two crops, almost each year of the 8-yr application.

Soil electrical conductivity (ECe) determined before and after the corn experiment was affected by fertilization treatments at the end of the experiment and by depth during both periods. In all cases, the highest ECe values were obtained for soil below 60 cm (Table 6). Annual manure application of 80 Mg ha–1 during the 4-yr corn experiment significantly increased soil salinity below 30 cm at the end of the experiment, and at levels similar to the inorganic fertilizers. However, in all cases the ECe values were lower than the critical limit (4 dS m–1) for most crop growth (Richards, 1954). In agreement with these findings, Evans et al. (1977) and Sutton et al. (1979) reported that soil EC significantly increased on application of liquid cattle manure at rates up to 336 Mg ha–1 yr–1, but remained at levels below the critical limit, even at the highest rate. However, high rates of liquid cattle manure are not suggested for continued annual application, due to the risk of plant injury associated with increased soil salinity (Evans et al., 1977). Values of ECe, measured in the 30-cm soil depth from organic or inorganic fertilization treatments during the corn experiment were only slightly higher than the initial ECe of 0.58 ± 0.05 dS m–1 reported by Matsi et al. (2003).


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Table 6. Soil electrical conductivity (ECe), in the beginning and at the end of the experiment at Thessaloniki, Greece.

 
Soil organic C and Kjeldahl N, at the end of the corn experiment, were not influenced by manure and remained at levels similar to those determined in the beginning of the experiment (Table 7). Organic C and Kjeldahl N content of manure plots during the corn experiment (Table 7) were similar or lower than initial values (7.5 ± 1.1 g kg–1 for soil organic C and 1.0 ± 0.1 g kg–1 for Kjeldahl N) reported by Matsi et al. (2003). Consequently, it seemed that both soil properties were not affected by the eight annual applications of the liquid dairy cattle manure. This is expected, since increases in soil organic C and total N have been associated with solid cattle manure (Chang et al., 1991; Eghball, 2002), due to its higher dry matter content in comparison to the liquid manure (Sutton et al., 1986). However, Culley et al. (1981) reported that soil organic C was significantly increased on application of liquid dairy cattle manure after 5 yr, but at a rate almost five times the rate used in this study.


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Table 7. Soil organic C and Kjeldahl N (0- to 30-cm depth), in the beginning and at the end of the experiment at Thessaloniki, Greece.

 
In conclusion, under a Mediterranean environment, soil incorporation of liquid dairy cattle manure at rates equivalent to inorganic fertilization can increase corn grain and silage yields and macronutrient concentration and uptake at levels similar to those of the inorganic fertilization. Long-term use of manure can improve soil fertility, with respect to soil-available NO3–N, P, and K; however, buildup of soil-available nutrients, organic C, and Kjeldahl N is not expected. In addition, soil salinity, due to long-term manure application, is expected to increase, but at levels acceptable for most crops and similar to levels caused by the inorganic fertilizer use.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 




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