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Published online 27 April 2005
Published in Agron J 97:791-798 (2005)
DOI: 10.2134/agronj2004.0207
© 2005 American Society of Agronomy
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Production Papers

Surface Application of Lime for Crop Grain Production Under a No-Till System

Eduardo F. Cairesa,*, Luís R. F. Alleonib, Michel A. Cambrib and Gabriel Bartha

a Universidade Estadual de Ponta Grossa (UEPG), Dep. of Soil Science and Agricultural Engineering, Av. Gen. Carlos Cavalvanti, 4748, 84030-900, Ponta Grossa, Paraná, Brazil
b Universidade de São Paulo (USP), College of Agriculture Luiz de Queiroz, Dep. of Soils and Plant Nutrition, P.O. Box. 9, 13418-900, Piracicaba, São Paulo, Brazil

* Corresponding author (efcaires{at}uepg.br)

Received for publication July 30, 2004.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The effectiveness of surface application of lime to soils under a no-till (NT) system, particularly with regard to subsoil acidity, is uncertain, and long-term data is needed to determine optimum surface liming rates in this cropping system. A field experiment was performed in the period from 1993 through 2003 in Paraná State, Brazil, on a loamy, kaolinitic, thermic Typic Hapludox to evaluate the extent of the downward movement of surface-applied lime in a NT system, and the effect on grain yields under crop rotation. The treatments consisted of dolomitic limestone at the rates of 0, 2, 4, and 6 t ha–1, calculated to raise the base saturation in the topsoil (0–20 cm) to 50, 70, and 90%. Surface-applied lime under NT was effective in alleviating soil acidity below the point of placement, and increased the cumulative grain yield of the crops. The effects of surface liming on all three acidity-related variables (pH, Al, and basic cations) were significant at 0- to 5- and 5- to 10-cm depths from 1 yr onward, and also at the 10- to 20-cm depth from 2.5 yr onward, remaining consistent for a period of up to 10 yr after liming. The maximum economic yield was obtained at 4 t ha–1 of limestone, showing that the lime rate estimated by the soil base saturation method at 70% in the 0- to 20-cm depth was appropriate for surface liming recommendation in a NT system.

Abbreviations: CEC, cation exchange capacity • NT, no-till • OM, organic matter


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
NO-TILL (NT) systems with diversified crop rotations have stood out as one of the most effective strategies to improve the sustainability of farming in tropical and subtropical regions, contributing to minimize soil and nutrient losses by erosion. In this cropping system, considerable increases in organic C and N contents take place, mainly in the top layers of the soil, as a result of plant residue deposition and absence of tillage. The increase in organic matter (OM) content brings about increases in cation exchange capacity (CEC) and biological activity. No-till has shown a rapid increase in cultivated areas in Brazil, currently taking up some 17 million ha.

Soil acidity limits agricultural yield in extensive areas in the world. Calcium deficiency (Ritchey et al., 1982) and Al toxicity (Pavan et al., 1982) are considered major yield-limiting factors of tropical and subtropical acid soils. Soil acidity problems are commonly corrected by applying limestone. To control soil acidity in NT, lime is broadcast on the surface without incorporation. The effectiveness of surface lime application to soils under NT, particularly with regard to subsoil acidity, is uncertain. Liming in general does not have an effect on reducing soil acidity beyond the point of placement, which depends on the leaching of salts throughout the soil profile. Sumner (1995) summarized many experiments where lime was surface applied. The review indicated that changes in the subsoil were slow with lime additions when no—or little—acidic N fertilizer was applied. Results of field studies show that the movement of lime to depth varies according to the timing and the rates of liming, soil type, weather conditions, management of acidic fertilizers, and cropping systems (Moschler et al., 1973; Blevins et al., 1978; Oliveira and Pavan, 1996; Rheinheimer et al., 2000; Gascho and Parker, 2001; Conyers et al., 2003). Even so, very little information is available on long-term surface lime application studies to control soil acidity under the NT system in Brazilian soils.

Several experiments conducted under diverse soil and climate conditions have attested the viability of surface liming, after the establishment of a NT system, for crop production (Moschler et al., 1973; Lal, 1976; Blevins et al., 1978; Hargrove et al., 1982; Grove and Blevins, 1988; Caires et al., 2000). However, NT affects some chemical characteristics related to soil acidity that may influence plant development. The higher content of OM (Bayer et al., 2000; Rhoton, 2000) and the greater concentration of P on the soil surface (Rhoton, 2000) under NT may reduce Al toxicity (Ernani et al., 2002). The rise in soil CEC due to the higher content of OM can provide sufficient concentrations of exchangeable cations, even in highly acidic soils (Caires et al., 1998). In addition, soil cover reduces water losses by evaporation and provides greater available moisture in the top layers, which may promote nutrient uptake under adverse acidic soil conditions (Caires and Fonseca, 2000). These effects raise doubts as to the optimum surface liming rates needed for providing high yields in this cropping system.

We evaluated the amelioration of soil acidity with time and the grain yields of crops in rotation under a NT system after surface application of lime rates to obtain a suitable method for surface liming recommendation in this cropping system.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The experiment was performed in Ponta Grossa, PR, Brazil (25°10' S, 50°05' W), on a Typic Hapludox. At the beginning of the experiment, soil chemical and granulometric analyses of the 0- to 20-cm depth showed the following results: pH (1:2.5 soil/0.01 mol L–1 CaCl2 suspension) of 4.5; exchangeable Al, Ca, Mg, and K contents of 6, 16, 10, and 1.4 mmolc dm–3, respectively; total acidity pH 7.0 (H + Al) of 58 mmolc dm–3; P (Mehlich-1) of 9.0 mg dm–3; total OM of 33 g dm–3; base saturation of 32%; and 295, 240, and 465 g kg–1 of clay, silt, and sand, respectively. The clay fraction had 265.8 g kg–1 of kaolinite (90.1%), 26.8 g kg–1 of goethite (9.1%), and 2.4 g kg–1 of hematite (0.8%). Before the establishment of the experiment, the field site had been used for grain cropping under the NT system during 15 yr.

A randomized complete block design was used and four treatments were replicated three times. Plot size was 8.0 by 6.3 m. The treatments consisted of dolomitic limestone at the rates of 0, 2, 4, and 6 t ha–1. The lime rates (LR) were calculated to raise the base saturation in the topsoil (0–20 cm) to 50, 70, and 90% as:

where V2 is base saturation estimated (50, 70, and 90%), and V1 is actual base saturation (soil analysis).


where Caex, Mgex, and Kex are exchangeable basic cations, and CEC is total cation exchange capacity.


ECCE is the effective calcium carbonate equivalent of the lime material. The dolomitic lime used had 84% of ECCE and was broadcast on the soil surface in July 1993. The lime was applied during the black oat (Avena strigosa Schreb.) growth, 4 mo before the first crop was sown.

Fertilizer rates varied with crops and years (Table 1), according to soil test recommendations for the State of Paraná. Nitrogen, P, and K were applied, most times, as urea, triple superphosphate, and potassium chloride, respectively. For the wheat (Triticum aestivum L.) crop in 2003, N was applied as ammonium nitrate. Black oat, mixed or not with common vetch (Vicia sativa L. subsp. sativa), was sown without fertilizers in May of each year and grown as a cover crop. Soybean [Glycine max (L.) Merr.] was sown each year in November at a seeding rate of 20 seeds m–1 (inoculated with Bradyrhizobium japonicum), and row spacing of 0.45 m. Corn (Zea mays L.) was always sown in October at a seeding rate of five seeds m–1, and row spacing of 0.90 m. Wheat and triticale (X Triticosecale) crops were sown in June at a seeding rate of 35 seeds m–1, and row spacing of 0.17 m. Grain was harvested from 13.5 m2, and grain yield was expressed at 130 g kg–1 moisture content. Plant residues were left on the soil surface following grain harvest. Weeds were controlled by appropriate herbicide treatments.


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  		Table 1. Cropping history and amounts of N, P, and K applied for 10 yr.

 
Throughout the development period of the crops grown during the spring–summer season (soybean and corn), there was no water limitation (Table 2). The crops grown during the autumn–winter season were more affected either by drought stress or excess of water. Grain yields of wheat in 1996 and triticale in 1997 were reduced by excessive rainfall occurred during the harvest. On the other hand, there was an extended water deficit during the vegetative stage and soon after the flowering of the wheat plants in 2003.


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  		Table 2. Seasonal rainfall.

 
Soil samples were taken from each plot at the following depths: 0 to 5, 5 to 10, and 10 to 20 cm. Twelve soil core samples per plot were taken by means of a soil probe sampler to obtain a composite sample. The samples were taken 1, 2.5, 5, 7.5, and 10 yr after the start of the liming treatments. Soil pH was determined in a 0.01 mol L–1 CaCl2 suspension (1:2.5 soil/solution). Exchangeable Al, Ca, and Mg were extracted with neutral 1 mol L–1 KCl in a 1:10 soil/solution ratio. Aluminum (KCl-exchangeable acidity) was determined by titration with 0.025 mol L–1 NaOH solution, and Ca and Mg by titration with 0.025 mol L–1 EDTA. All the soil chemical attributes were analyzed according to standard procedures used by the Agronomic Institute of the State of Paraná (Pavan et al., 1992).

The data from the soil chemical analysis and crop grain yields was analyzed through analysis of variance and simple regression methods, using the SAS reference (SAS Institute, 1985).


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Amendment Effects on Soil Chemical Attributes
Surface liming raised pH and reduced the contents of exchangeable Al in the soil (Fig. 1); the magnitude of the alterations varied according to the lime rates applied, the reaction time of the neutralizing agent, and the soil depth. Surface-applied lime brought about a more marked improvement in acidity in the surface layer of the soil (0–5 cm) and, to a lesser degree, at 5- to 10- and 10- to 20-cm depths. The effects of the application of lime rates on both pH and exchangeable Al were significant at 0- to 5- and 5- to 10-cm depths at 1 yr following liming, and also at the 10- to 20-cm depth at 2.5 yr after liming, and remained consistent for a period of up to 10 yr after liming. The increase in pH with the highest lime rate applied, during the evaluation period, ranged from 1 to 1.7 pH at the 0- to 5-cm depth, and from 0.7 to 1.3 pH at the 5- to 10-cm depth. The highest increase in pH for these two depths occurred at 2.5 yr after liming. The raise in pH at the 10- to 20-cm depth varied only from 0.2 to 0.4 pH with the application of the highest lime rate. Exchangeable Al contents in the plots without lime were low (<5 mmolc dm–3) in the uppermost layers of the soil (0–5 and 5–10 cm) up to 2.5 yr after the application of the treatments, but after that period increased to levels that are considered toxic (5–10 mmolc dm–3). At the 10- to 20-cm depth, exchangeable Al levels in the plots without lime were high since the beginning of the experiment.



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  		Fig. 1. Changes in soil pH 0.01 mol L–1 CaCl2 and exchangeable Al (Alex) for the depths of (•) 0–5 cm, ({blacksquare}) 5–10 cm, and ({blacktriangleup}) 10–20 cm at (a) 1, (b) 2.5, (c) 5, (d) 7.5, and (e) 10 yr after surface liming rates application. *P < 0.05; **P < 0.01.

 
The changes in soil exchangeable Ca and Mg levels as a result of the liming rates (Fig. 2) practically followed the changes in pH and exchangeable Al (Fig. 1). Changes in pH alter the surface chemistry of colloids because of the variable pH nature of the surface charges in Brazilian soils (van Raij and Peech, 1972). Increasing inputs of acids in these soils result in the depletion of basic cations and in the release of Al. On the other hand, the elevation in soil pH through liming increases basic cation retention due to the increase in negative variable electric charges that are generated on the surface of colloids by the dissociation of H+ from the hydroxyl groups. The effects of the application of lime rates on the contents of exchangeable Ca and Mg were significant at the 0- to 5- and 5- to 10-cm depths at 1 yr after liming. There was an increase in exchangeable Ca levels at the 10- to 20-cm depth only at 2.5 yr after liming. It took between 2.5 and 5 yr after liming for an increase to take place in the contents of exchangeable Mg at 10 to 20 cm. Such effects remained consistent for a period of up to 10 yr after liming.



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  		Fig. 2. Changes in exchangeable Ca and Mg (Caex and Mgex) for the depths of (•) 0–5 cm, ({blacksquare}) 5–10 cm, and ({blacktriangleup}) 10–20 cm at (a) 1, (b) 2.5, (c) 5, (d) 7.5, and (e) 10 yr after surface liming rates application. *P < 0.05; **P < 0.01.

 
Soil acidification was little in the no-lime plots during the evaluation period (Fig. 3). There was a significant decrease (P < 0.01) in soil pH (y) as a function of time (x, in years) only at the 0- to 5-cm depth (y = 4.59 – 0.04x, R2 = 0.60). This surface layer acidification in NT is normally caused by the decomposition of plant residues and by the reaction of N fertilizers on the surface. Because of the predominance of soybean in the crop rotation, low rates of N were applied (Table 1). There was no soil acidification at the 5- to 10-cm depth, and a significant slight increase was observed (P < 0.01) in soil pH (y) as a function of time (x, in years) at the 10- to 20-cm depth (y = 4.02 + 0.02x, R2 = 0.44). In the 5- to 10- and 10- to 20-cm layers, the dissolution of Al minerals in the no-lime plots must have buffered the effect of acids originated from the C and N cycles in the pH range between 4.0 and 4.3. The magnitude of the alterations in soil pH was positively correlated to the lime rates, but the velocity of lime reaction was similar for the different rates. There was an increase in pH at 10 to 20 cm only after the lime had reached its maximum reaction, depending on the applied rate, in the uppermost soil layers. In order for lime applied on the surface to have an influence on subsoil acidity, alkalinity usually in the form of HCO3– or OH– must be transported downward by mass flow from surface layers (Sumner, 1995). It so happens that anions HCO3– and OH–, originated from the lime dissolution, react with acidic cations from the soil solution (H+, Al3+, Mn2+, and Fe2+). As long as these acidic cations exist, the acidity neutralization reaction will be limited to the surface layer of the soil, slowing the effect at the subsurface level. This explains why there was a significant alkalinity movement of surface-applied lime to the 10- to 20-cm depth only when pH (0.01 mol L–1 CaCl2) in the uppermost layers reached values above 5.0 (Fig. 3). Although the loss of HCO3– and OH– from the surface layers (0–5 and 5–10 cm) renders these layers more acidic, the addition of those ions to the underlying layer (10–20 cm) represents an alkaline addition.



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  		Fig. 3. Effect of the time after surface-applied lime at the rates of ({circ}) 0, (•) 2, ({blacktriangleup}) 4, and ({blacksquare}) 6 t ha–1 on soil pH 0.01 mol L–1 CaCl2, considering the depths of (a) 0–5 cm, (b) 5–10 cm, and (c) 10–20 cm.

 
Field research has shown seemingly discordant results as to the movement of surface-applied lime in a NT system, which may be related to liming rates, time of sampling after lime addition, soil type, climate conditions, management of acidic fertilizers, and cropping systems. In a long-term trial conducted in southeastern Australia, on a soil with 290 g kg–1 of clay and 22 g kg–1 of OM, Conyers et al. (2003) found that the application of 1.5 t ha–1 of high-quality limestone (98% CaCO3, 99.5% <250 µm diam.) on the surface took from 2 to 4 yr to reach the depth of 10 cm and was not effective in raising pH below that depth for a period of 8 yr. This greater resistance to alkaline movement in Australian soil may be related to the low lime rate used (enough to neutralize exchangeable Al in the 0- to 10-cm soil layer) associated to an average rainfall of only 570 mm yr–1. To enable the effective use of limestone in a NT system, this study proposed applying limestone at a higher rate than calculated or measured for the site where limestone was to be incorporated, so as to drive the leaching of bicarbonate. In a trial conducted in southern Brazil (Rheinheimer et al., 2000) in a soil with 37 g dm–3 of OM and 33 mmolc dm–3 of exchangeable Al, it was found that surface-applying lime at high rates (17 t ha–1) created an alkalinization front which advanced in depth with time, but only reached the depth of 10 cm after 4 yr. In this study, however, the trial area had been managed as native pastureland before the establishment of NT. In the USA, the increase in soil pH at the 10- to 20-cm depth after liming during 8 yr was from 0.2 to 1.0 pH under NT (Moschler et al., 1973). These results contrast with ours, probably due to the limestone being applied annually in this study. In another trial conducted in southern Brazil, under similar climate conditions to our study (Oliveira and Pavan, 1996), on a soil with 620 g kg–1 of clay and 46 g kg–1 of OM, it was found that the increase in soil pH at the 10- to 20-cm depth with surface application of lime (5.5 t ha–1), during 32 mo, was from 0.5 to 1.8 pH under NT. These results contrast strongly with ours, but in this study the soil had been cultivated for decades under the conventional tillage system before the establishment of the study using NT system.

Crop Grain Yield
Soybean grain yield was hardly influenced by the surface-applied lime rates (Fig. 4). Out of the eight soybean harvests, grain yield was only increased by liming in 1998–1999. In that agricultural year, the response was linear and the average increase in grain yield was on the order of 10% with the highest lime rate applied (6 t ha–1). The average soybean yield of the trial throughout the eight harvests was 3.18 t ha–1. These results differ substantially from those obtained by Oliveira and Pavan (1996), who found increase in grain yield of 42% with surface application of lime for four consecutive soybean crops under NT. The discrepancy in the results can be explained by the higher level of exchangeable Al (8.5 mmolc dm–3) and the lower level of exchangeable Mg (6.6 mmolc dm–3) in the surface layer of the soil studied by Oliveira and Pavan (1996), as compared with this study. Assessments of the effect of liming on crop yield for 31 yr on a loamy sand soil with low CEC (fine-loamy, siliceous, thermic Plinthic Kandiudult) of the Georgia Coastal Plain (USA) showed no beneficial effect from lime for soybean yields in 9 of 11 yr (Gascho and Parker, 2001), in agreement with the results obtained in the present study.



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  		Fig. 4. Crop yield of soybean, corn, and wheat or triticale as affected by surface liming rates application. *P < 0.05; **P < 0.01.

 
Corn grain yield in 1994–1995 and 2000–2001, wheat in 1996, and triticale in 1997 were not influenced by liming (Fig. 4). The average yield of the two corn harvests of the experiment was 9.65 t ha–1 of grain. Wheat yield in 1996 and that of triticale in 1997 were impaired by the excessive rainfall that occurred during the harvest season (Table 2). Wheat yield in 2003 was raised quadractically with the application of lime rates. According to the adjusted regression equation, the maximum wheat yield in that year would be achieved with the rate of 4.5 t ha–1 of lime, providing an increase in yield on the order of 2.5 t ha–1 of grain. This marked response of wheat to liming was probably related to rainfall distribution during the development stages of the crop (Table 2). Although the rain episodes occurred in adequate intensity soon after sowing and at the beginning of wheat flowering, rainfall was irregular during the crop cycle—extended water deficit was observed during the vegetative stage and soon after flowering. Since the wheat plants grown in the plots without lime revealed chlorosis and growth problems, especially during the drought periods that occurred in the vegetative stage, it is possible that the toxicity of Al for the plants has shown more intensely when there was less moisture in the soil.

High crop grain yields were probably obtained in the plots without lime (Fig. 4) because OM (Bayer et al., 2000; Rhoton, 2000) and P (Rhoton, 2000) accumulate in the upper few centimeters under NT soil, which may reduce Al toxicity (Ernani et al., 2002). However, this effect should only have importance in the absence of water deficit (Table 2). Crop response to liming under NT due to decreases or elimination of Al phytotoxicity seems to be quite dependent on the water regime occurring during the growth cycle of the plants.

The cumulative grain yield of the crops, in the period from 1993 through 2003, increased with the application of lime rates on the soil surface, exhibiting a quadractic response (Fig. 5). Based on the regression equation obtained, the maximum yield would be reached with the rate of 4.2 t ha–1 of lime and the average increase in grain yield would be on the order of 5.1 t ha–1 (11%). Yield at 6 t ha–1 of lime was slightly lower likely by the presence of higher soil pH values in surface layers (Fig. 1). Caires and Fonseca (2000) found that a increase in pH (0.01 mol L–1 CaCl2) from 5.4 to 5.8 at the 0- to 5-cm depth, and from 4.8 to 5.1 at 5- to 10-cm depth, by surface liming under NT, decreased soybean uptake of Zn and Mn in 24 and 33%, respectively. A single wheat harvest in 2003 accounted for about 50% of that increase in the cumulative grain yield as a result of liming. The sum of the harvests of the other cereal crops (corn in 1994–1995 and 2000–2001, wheat in 1996 and triticale in 1997) contributed with 30%, and soybean, in the eight-harvest sum, with 20%, approximately, to this increase in cumulative grain yield. A lower soybean suscetibility to Al than that of cereal crops might be seen as a possible explanation for these results. In a trial conducted in a highly acidic soil with 28 g dm–3 of OM, soybean showed to be more susceptible to Mn toxicity than to Al, whereas the inverse was observed for sorghum (Gallo et al., 1986). In addition, since crop response to lime occurred only when substantial amounts of N were applied, Gascho and Parker (2001) found no beneficial effect from lime for leguminous crops yields.



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  		Fig. 5. Influence of surface-applied lime rates on cumulative grain yield in the period from 1993 through 2003. Crop rotation: soybean, corn, soybean, wheat, soybean, triticale, soybean, soybean, soybean, corn, soybean, soybean, and wheat. *P < 0.05.

 
Adopting the criterion of unlimited capital, amortization throughout the experiment period, and prices at $15.00 and $137.60 t–1 for limestone and grain, respectively, the maximum economic yield was obtained with 4 t ha–1 of lime. The price of grain was calculated by the weighed average of the products from the 13 harvests, considering the following prices per ton: $163.00 for soybean, $88.30 for corn, $116.70 for wheat and $75.0 for triticale. The economic returns of liming, on a 10-yr average, was on the order of $60 ha–1 yr–1, which is in line with the results by Gascho and Parker (2001). The lime rate calculated to achieve maximum economic yield was the one recommended by the soil base saturation method at 70%, for a sample collected at the 0- to 20-cm depth, indicating that this is an adequate criterion for surface liming recommendation under NT system.


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Surface-applied lime under a NT system was effective in alleviating soil acidity below the point of placement. However, there was only an increase in pH at the 10- to 20-cm depth after the lime had reached its maximum reaction, depending on the applied rate, in the uppermost layers of the soil (0–5 and 5–10 cm). This shows that, even though the loss of HCO3– and OH– from the surface layers renders these layers more acidic, the addition of those ions to the layer below represents an alkaline addition. Nevertheless, the increase in pH at 10 to 20 cm ranged only from 0.2 to 0.4 pH with the application of lime on the surface.

Surface liming increased grain yield in only two crops (one of soybean and one of wheat) of the 13 harvests evaluated (eight of soybean, two of corn, two of wheat, and one of triticale). The cumulative grain yield of the crops increased in 5.1 t ha–1 (11%) with liming, and the maximum economic yield was obtained with 4 t ha–1 of lime. A single wheat harvest accounted for about 50% of that increase in cumulative grain yield, probably as a result of extended water deficit during the vegetative stage and soon after flowering, showing that the response of the crops to liming under NT is dependent on the water regime during the growing cycle of the plants. The lime rate calculated to achieve maximum economic yield was the one recommended by the soil base saturation method at 70%, for a sample collected at the 0- to 20-cm depth, indicating that this is an adequate criterion for surface liming recommendation under NT system.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Research supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), a Brazilian agency related to scientific development.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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