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USDA-ARS, 1991 Upper Buford Circle, Room 439, St. Paul, MN 55108-6028
* Corresponding author (crosen{at}soils.umn.edu)
Received for publication February 11, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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25 mm drainage water in at least one 24-h period) and excessive irrigation in the third year, PCU at 280 kg N ha-1 improved total and marketable tuber yields by 12 to 19% compared with three applications of urea. Fertilizer N RE estimated by the difference and 15N isotope methods at the 280 kg N ha-1 rate was, on average, higher with PCU (mean 50%) than urea (mean 43%). Fertilizer N RE values estimated by the isotope method (mean 51%) were greater than those estimated by the difference method (mean 47%). Results from this study indicate that PCU can reduce leaching and improve N recovery and tuber yield during seasons with high leaching.
Abbreviations: CRF, controlled-release fertilizer • DAP, days after planting • PCU, polyolefin-coated urea • RE, recovery efficiency
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Twelve percent of public wells in major potato-growing counties in Minnesota during 1997–1998 were above the USEPA 10 mg L-1 limit for NO3–N (Gallus and Montgomery, 1998). Elevated NO3 levels have also been measured in ground water sources adjacent to potato fields in Wisconsin (Saffigna and Keeney, 1977) and southern Ontario, Canada (Hill, 1986). In Quebec, NO3–N concentrations of up to 40 mg L-1 have been measured in subsurface water draining from a sandy field cropped to potato (Madramootoo et al., 1992). Mass balance data from the study by Hill (1986) indicated that 78 to 220 kg N ha-1 was lost annually below 183 cm under potato crops receiving 160 to 210 kg N ha-1 annually.
Nitrate accumulation in the soil and leaching out of the root zone are determined by, among other things, the amount of N applied relative to crop demand and removal. Nitrate leaching can therefore be minimized by either reducing N fertilizer rates or increasing the proportion of applied N removed in the harvested portion of the crop. Nitrogen loss from fertilizer applications results in lower N RE in crop production. Conversely, low recovery of applied N by the crop can augment N loss to the environment. Results from a study by Gerwing et al. (1979) in central Minnesota confirmed that maintaining high N RE would minimize the amount of N subject to leaching. In potato production, fertilizer N recoveries commonly range between 30 and 70% (Hill, 1986; Errebhi et al., 1998; Meyer and Marcum, 1998).
Past potato fertility research on irrigated sandy soils has focused mainly on irrigation management and N fertilizer rates, placement, and timing (Errebhi et al., 1998; Waddell et al., 2000). However, even with properly timed season-long N management and appropriate irrigation plans, controlling NO3 leaching is difficult due to unforeseen rainfall events immediately following irrigation or N application (Sexton et al., 1996). Therefore, more effective alternative approaches to reduce NO3 loss to ground water are needed.
Controlled-release fertilizers (CRFs) may be one such alternative that may improve N recovery by the crop, thereby minimizing excessive NO3 leaching. Nitrogen release from traditional products, such as sulfur-coated urea, has been unpredictable (Trenkel, 1997). Recently, improved CRFs have been developed with polymer coating technology to modify the rate and duration of nutrient release. Polymer-coated CRFs can improve N use efficiency in corn (Zea mays L.) (Shoji et al., 1991) and decrease NO3 leaching (Wang and Alva, 1996). Zvomuya and Rosen (2001) reported higher potato yields for PCU compared with urea, but effects on N leaching and N RE were not evaluated.
The objectives of this study were to (i) investigate the efficacy of PCU as a tool in minimizing NO3 leaching and improving N RE in potato and (ii) characterize the fate of PCU- and urea-N applied to irrigated potatoes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Before planting, the experimental area received broadcast applications of 225 kg ha-1 potassium chloride and the same rate of potassium–magnesium sulfate, which were immediately incorporated. Triple superphosphate and additional potassium–magnesium sulfate were applied at planting in a double band 7 cm to the side and 5 cm below each seed piece to achieve total applications of 55 kg P ha-1, 166 kg K ha-1, 25 kg Mg ha-1, and 45 kg S ha-1.
The test cultivar was Russet Burbank, the most popular potato used for processing in the upper Midwest. Cut seed pieces (55–85 g) were hand-planted in open furrows the third week of April each year. Spacing was 90 cm between rows and about 25 cm between seed pieces in the row. Agrochemicals for the control of pests, diseases, and weeds were applied as needed with standard practice recommended for the crop in the area (Hutchison, 1996).
Soil moisture deficits of 25 mm or less during the vegetative and maturity stages and 14 mm or less during tuber bulking were maintained in the root zone (0–60 cm) with supplementary irrigation supplied according to the checkbook method (Wright and Bergsrud, 1991). Meteorological data were recorded hourly at a weather station within 1 km of the experimental plots.
To ensure greater leaching than occurred in the first two years of the study, an extra experiment (1999a) with the same N treatments was established in the same field in 1999 to receive irrigation based on early season rainfall events from a severe leaching year (Errebhi et al., 1998). Three times the amount of water applied in the standard-irrigation experiment was applied during the first three irrigation events in 1999. Subsequent irrigation was the same for the two irrigation regimes.
Two N sources, urea and a 70-d release formulation of PCU (Chisso Co., Tokyo, Japan1), were compared at 140 and 280 kg N ha-1. The lower rate was the optimum obtained for PCU in a previous study (Zvomuya and Rosen, 2001), whereas the higher rate was based on prevailing fertilization practices of potato growers in central Minnesota (Bruening and Montgomery, 1995). Control plots that received no N were included in each replication. All PCU N was supplied in a single application at planting. Urea treatments received an initial 28 kg N ha-1 at planting, with the remaining N added in equal applications at emergence (28–30 days after planting, DAP) and hilling (37–45 DAP). Nitrogen applied at planting was banded together with the basal fertilizers during furrow opening. Application at emergence involved N addition in a double band 2.5 cm deep and 20 cm to each side of the plant row. At hilling, urea was surface-applied on either side of the hill and then incorporated into the hill within 6 h. An additional urea treatment (posthill) in 1998 and 1999 included 90 of the 280 kg N ha-1 as a 1:1 mixture of urea and ammonium nitrate applied in two additional splits 21 and 35 d after hilling. Posthill N was broadcast by hand on the crop canopy, followed immediately by irrigation to minimize urea N loss through volatilization. Many farmers in the region apply some of the N requirement of the potato crop as urea–ammonium nitrate (UAN) in irrigation water after hilling.
Treatments were replicated four times and arranged in a randomized complete block design. Each plot was 6 m in length in all years and either four (1997) or six rows wide (1998 and 1999). The change in plot width after 1997 was necessary to accommodate the extra row of 15N-treated plants as described below.
The 15N-labeled urea (5 atom %) and PCU (3.2 atom %) were applied in four replications in 1998 and three replications in 1999 to a 1.5-m section of an inner nonharvest row within the experimental plots. At planting, the preweighed labeled fertilizer was hand-applied in a double band at the same time nonlabeled fertilizer was applied, except that the posthill treatment received no labeled fertilizer.
Soil temperature at the depth of the fertilizer band was recorded every 30 min with two Optic StowAway loggers (Onset Computer Corp., Bourne, MA) installed in each of three replications. Soil moisture content at the same depth was measured with two CS615 water content reflectometers (Campbell Scientific, Logan, UT) connected to a CR10 datalogger that recorded measurements every 30 min.
Dissolution of Polyolefin-Coated Urea
In situ field incubation of PCU was undertaken in each experiment to characterize dissolution of the fertilizer. Eight to ten plastic mesh bags containing 3 g of fertilizer and 5 g of soil from the experimental field were buried within the fertilizer band at planting in nonharvest rows of appropriate plots in three replications. One mesh bag from each replication was retrieved every 2 to 3 wk for determination of total N. Dissolution of PCU as a function of time, accumulated mean soil temperature (AST), or growing degree days (GDD) was determined by regression. Accumulated mean soil temperature was calculated by summing mean daily soil temperatures recorded since the day of fertilizer application. Growing degree days were computed by subtracting 5°C from the mean daily soil temperatures, followed by summation of the adjusted mean daily soil temperatures. The rationale for this computation is that dissolution of PCU is limited if temperature falls below the threshold value of 5°C (Dr. Sadao Shoji, personal communication, 2001).
Estimation of Nitrate Nitrogen Leaching
Soil water was extracted with suction lysimeters with porous ceramic cups of 0.1 MPa air-entry pressure (Soilmoisture Equipment Corp., Santa Barbara, CA). The suction lysimeters were installed to a depth of 120 cm in the hill of a harvest row in 1997 and in the hill and furrow of a nonharvest row in 1998 and 1999. Samplers were installed in each plot (one lysimeter per plot in 1997 and two per plot in 1998 and 1999) in three replications within a week after planting. The samplers were installed vertically into slightly larger holes into which about 250 mL of silica flour had been added to improve hydraulic contact between the ceramic cup and the surrounding soil. Soil augured out during hole preparation was repacked into the hole after insertion of the samplers. The samplers were sealed with bentonite near the soil surface to minimize water flow along the shaft. Using a hand pump, a suction of 40 kPa was applied to collect samples from the soil water draining through the soil at the depth of installation. The 40-kPa vacuum was considered sufficient to maintain the suction in the cup above that of the surrounding soil until sample extraction. Solutions were extracted from the samplers with a hand pump at weekly or shorter intervals starting about 3 to 4 wk after planting. Samples were stored frozen until analysis. Nitrate and NH+4 in the soil solutions were determined by the diffusion–conductivity method described by Carlson et al. (1990).
After harvest of the potato crop, solution sampling tubing was buried beneath the Ap horizon to permit normal tillage operations and seeding of winter rye. The tubing was retrieved immediately after sowing and the disturbed areas around them resown.
Deep Percolation
Deep percolation under the potato crop was estimated on a daily basis at a depth of 120 cm with a simplified water balance equation (Waddell et al., 2000). The 120-cm depth was chosen to ensure that measurements were taken well below the root zone. The water balance between two consecutive days, i and j, was calculated as:
 | [1] |
S was change in soil water storage between two consecutive days, and E was evapotranspiration. The E values were calculated as a product of the crop coefficient (Kc) at a given crop development stage and potential evapotranspiration (Eo) estimated by a modified Penman equation (Wright and Bergsrud, 1991) with daily weather data recorded at the experimental site. Maximum water storage on any given day was equal to the soil water holding capacity (SWC) of the 120-cm soil profile. Beginning in early spring, the soil profile was set at SWC. Nitrate leached in each sampling interval was calculated by multiplying NO3–N concentration in the soil solution at successive sampling dates by the amount of drainage between the sampling dates. Although this method of calculation may not take into consideration daily fluctuations of NO3–N concentration, possible errors were minimized by maintaining a continuous vacuum in the suction lysimeters and by sampling at short intervals (7 d or less). In 1998 and 1999, leaching losses from each plot were obtained by averaging the losses estimated under the hill (inrow) and under the furrow (between rows). Nitrate leaching for 1997 was calculated from inrow samplers only because no between-row sampling was done that year. Results from the three experiments in 1998 and 1999 indicated that at low to medium leaching levels, such as those obtained in the greater part of 1997, differences between inrow and between-row losses were small. Mean leaching losses from each treatment were summed over the growing season to give total N loss.
Harvesting and Plant Tissue Analysis
Before plot harvest, vines from the central two harvest rows and tubers and vines from the middle four plants in the 15N microplots were harvested manually and weighed about 2 wk before tuber harvest. Potato tubers were mechanically harvested from the harvest rows during the third week in September each year. The tubers were graded into three size categories: <85 g, >85 g (marketable yield), and >170 g. Samples consisting of 25 potato tubers greater than 85 g were collected from each plot for determination of dry mass and measurement of specific gravity by the hydrometer method (Snack Food Association, 1995).
Tuber and vine samples from 15N-labeled and unlabeled treatments were dried at 60°C and weighed for dry matter yield. Ground samples were analyzed for total N with a modified Kjeldahl method in which NO3 is reduced by salicylic acid (Bremner, 1996). Ammonium in digests was determined conductimetrically (Carlson et al., 1990). Total N uptake was calculated as the product of total N concentration in plant tissue and dry matter yield. Atom % 15N in plant samples from 15N microplots and zero N plots was determined by spectrometric analysis with a Europa Scientific Integra isotope ratio mass spectrometer (PDZ Europa, Cheshire, UK) at the University of California–Davis Stable Isotope Facility.
Soil Sampling and Analysis
Six 2.5-cm-diameter soil cores were collected to a depth of 60 cm in the main plots immediately after harvesting. Cores from each plot were bulked and a subsample was taken for inorganic N determination. Air-dry soil samples were extracted with 2 M KCl and analyzed for residual NO3–N (Carlson et al., 1990). Bulk densities measured from the study site (Gremy et al., 1993) were used to calculate the amount of NO3–N in each plot.
Fertilizer Nitrogen Recovery Efficiency
The amount of N absorbed by tubers plus vines per kilogram of applied N (RE) was calculated based on 15N (REisot) uptake by the crop and by the nonisotopic difference method (REdiff). In the isotope method, RE was estimated from the 15N enrichment measurements with the amount of fertilizer N applied (NF) and the total N uptake by the crop (NP), both expressed in kg N ha-1:
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Calculation of recovery efficiency by the difference method was based on N uptake in control (N0) and fertilized plots (NFP), and the amount of fertilizer N applied (NF):
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An assumption of the difference method is that absorption of nonfertilizer N from the soil is the same for fertilized and control plants.
Statistical Analysis
Data from the study were analyzed with PROC GLM (SAS Institute, 1996). For the leaching data, analysis of variance was performed only on the total NO3–N loss over the entire growing season. Data expressed as percentages were log-transformed before analysis to achieve normal distribution or homogeneity of variance. Data for each year were analyzed separately because treatments were expanded after 1997 and because preliminary analysis of variance on the pooled data indicated significant year by treatment interactions for the independent variables measured. Treatment means were compared using the Waller–Duncan test using a k ratio of 50, which corresponds to the 10% probability of making a Type I error (Steel and Torrie, 1997). For the dissolution study, slopes of the regression curves of cumulative nitrogen release (CNR) were compared using PROC GLM (SAS Institute, 1996).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Dissolution of Polyolefin-Coated Urea
Cumulative N release was a quadratic function of time. Comparison of slopes of the regressions indicated that the quadratic functions were similar for the four experiments (p > 0.1). Dissolution of PCU was therefore described by a single quadratic equation with an x intercept of 14 d after fertilization (Fig. 1)
. The regression suggests that 60% of applied PCU N, or 84 and 168 kg N ha-1 for the 140 and 280 kg N ha-1 rates, respectively, was released by harvest time (about 150 d after application). There was no measurable N release during the first 2 wk after fertilizer application.
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Nitrate Leaching
In all experiments, NO3 leaching was highest following major rainfall and irrigation episodes (Fig. 3)
. At least 75% of the leaching losses from all treatments in 1997 occurred during the first three weeks in July (67–89 DAP). In 1998, only two leaching episodes with drainage volumes above 20 mm were observed at 66 (24 mm) and 84 DAP (35 mm). In the standard irrigation experiment in 1999, nearly one half of the total seasonal losses for each treatment occurred during the two sampling intervals ending 62 and 97 DAP. More than 40% of total seasonal losses in 1999 under excessive irrigation occurred between 43 and 62 DAP.
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Water sampling continued until freezing in November (about 200 DAP) each year and resumed the following spring under the succeeding rye crop. No measurable leaching occurred between harvest and freezing in any experiment due to dry conditions. Similarly, in the absence of irrigation and leaching rainfall, no significant percolation occurred in the subsequent year when the plots were cropped with rye. However, NO3–N concentrations, averaged across experiments and N rates, in water samples collected until harvest of the rye crop in mid-July each year were highest in plots previously fertilized with PCU (mean 6.9 mg L-1) and averaged 3.2 mg L-1 for urea and the control. Although all concentrations were below the 10 mg L-1 limit, it is apparent that this limit could be exceeded if the plots were to remain fallow the year following PCU application. These results indicate the importance of a subsequent crop in lowering the risk of NO3 leaching in potato fields that have received PCU N, or of selecting PCU that has a faster dissolution rate in this environment.
Dry Matter Yield
Tuber, vine, and total dry matter (DM) yields increased with N application in all experiments except 1998, when vine DM did not respond to low N application regardless of source (Table 2). Urea and PCU supported similar DM yields, except in 1997 when vine DM at 140 kg N ha-1 was higher with urea, whereas PCU gave higher total DM yield at the 280 kg ha-1 rate. Total DM with five split applications was less than total DM with PCU or urea in three split applications in 1998 and 1999 under standard irrigation.
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Tuber Yield
All N treatments significantly improved total and marketable tuber yield compared with the zero N control in all experiments, with the exception of the 140 kg N ha-1 urea treatment in 1998, which produced similar total yield as the control (Table 2). Total and marketable yields were higher with PCU than with three applications of urea at both the 140 and 280 kg N ha-1 rates in 1997 and at 280 kg N ha-1 under excessive irrigation in 1999, but the difference between the two sources at equivalent rates was not significant in 1998 and under standard irrigation in 1999. There was no benefit in total and marketable yield from applying urea N in five compared with three splits in any of the experiments, but PCU at 280 kg N ha-1 resulted in higher marketable yield in 1998 and higher total and marketable yields under excessive irrigation in 1999 than five applications of urea. Doubling the N rate from 140 to 280 kg ha-1 resulted in higher marketable yield for both sources in 1998 and higher total and marketable yields for PCU under excessive irrigation in 1999. The percentage of marketable tubers greater than 170 g, which are desirable for processing, was similar or higher for PCU than urea. Doubling the N rate from 140 to 280 kg ha-1 resulted in a similar or greater percentage of marketable tubers in this category.
Differences in yield between the two N sources in 1997 and under excessive irrigation in 1999 could be due to high and, in the case of the latter experiment, earlier leaching, which resulted in greater NO3 loss compared with 1998 and 1999 under standard irrigation, as discussed below. Westermann and Kleinkopf (1985) noted that decreased N uptake resulting from depleted soil NO3–N can reduce tuber bulking rates, size, and yields. Because PCU releases N slowly, N loss through leaching is minimized, resulting in higher yields and larger tubers compared with urea. It has also been reported that the benefits of CRFs relative to soluble fertilizers in potato production are associated with the continued supply of N during tuber bulking and earlier tuber initiation (Cox and Addiscott, 1976).
The present results corroborate earlier findings by Zvomuya and Rosen (2001). These authors obtained higher yields and larger tubers with a 1:1 blend of 50- and 70-d PCU formulations than three applications of urea during leaching seasons. In a majority of studies, traditional CRFs have resulted in lower potato yields than soluble fertilizers (Lorenz et al., 1972; Cox and Addiscott, 1976; Waddell et al., 1999). Poor performance of the CRFs in these studies was mostly due to unpredictable release of N, which did not match crop demand.
The absence of source or rate effects on total and marketable yields in the standard irrigation experiment in 1999 may have been due in part to the effect of the previous soybean crop. Potato yield response to N fertilization is often limited following a legume crop (Bélanger et al., 2000) due to release of N through mineralization during the growing season. Tuber size, however, increased with increased N even when total and marketable yields were not affected in our experiment.
Nitrogen Recovery
Fertilizer N generally increased tuber, vine, and total N uptake compared with the control in all experiments, with a few exceptions (Table 3). Total and tuber N uptake increased in all experiments as PCU N rate doubled from 140 to 280 kg ha-1. Except in 1998, N uptake from PCU by the vines responded similarly. In contrast, doubling the N rate as urea increased total, tuber, and vine N uptake only in 1998 and under standard irrigation in 1999. At 280 kg N ha-1, N recovery in the tubers, vines, and tubers plus vines (total) was 45% higher with PCU than urea application in 1997 and 23% higher under excessive irrigation in 1999. In 1998 and 1999 (standard irrigation), recoveries were similar for PCU and urea at the high N rate. The posthill treatment resulted in lower total N recovery than the equivalent rate of PCU N in 1999 under excessive leaching and than PCU and urea under standard irrigation in 1999. In 1998, total N uptake at each N rate was similar among N sources and managements, reflecting the lower leaching during that season. At the 140 kg N ha-1 rate, N uptake by tubers, vines, and the whole plant was similar for the two sources, except in 1997 when tuber N uptake was higher with PCU and vine N uptake was higher with urea.
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Nitrogen RE for PCU estimated by the difference method was 93% higher in 1997 and 54% higher under excessive irrigation in 1999 than urea at 280 kg N ha-1 (Table 3). The posthill treatment resulted in similar N RE estimates as PCU and three urea applications in 1998 and in both experiments in 1999. Recovery efficiencies estimated using the isotope method (REisot) were higher for PCU than urea at the 140 kg N ha-1 rate in all three experiments and at the 280 kg N ha-1 rate under excessive irrigation in 1999. Results for the higher N rate follow a pattern similar to those obtained for the percentage of total plant N derived from fertilizer (total Ndff) (Table 4). However, total Ndff was significantly higher with PCU than urea only in 1999 under excessive irrigation at the high N rate, with PCU out-performing urea by 13%. At 140 kg N ha-1, total Ndff was similar for urea and PCU N in all three experiments. The percentage of tuber N derived from fertilizer (tuber Ndff) was higher with PCU than urea in 1999 at both rates in the excessive irrigation experiment and at the higher rate under standard irrigation. Vine Ndff was higher with PCU in 1999 under excessive irrigation. Because the posthill treatment was not labeled, comparisons with other treatments cannot be made using the isotope method.
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The low recovery of urea N, particularly at the high rate under excessive irrigation in 1999, reflects the large leaching losses of N that occurred early in the season. Under the low leaching conditions in 1998, recovery of applied N was similar for PCU and soluble N treatments. Using the difference method, Errebhi et al. (1998) reported recoveries averaging 33% during a leaching season and 56% during a nonleaching year for Russet Burbank potatoes fertilized with 270 kg soluble N ha-1 on a similar soil. In other studies, recoveries of 50 to 60% have been reported for Russet Burbank potatoes fertilized with soluble N fertilizers (Joern and Vitosh, 1995). Based on the dissolution rate of the PCU and at the recommended rate of 280 kg N ha-1, the REdiff values obtained in this study translate to recoveries of 80, 64, and 91% of released N for 1997, 1998, and 1999, respectively. Corresponding recoveries of released N using the REisot method were 67% and 98% for 1998 and 1999, respectively.
The isotope method tended to be more precise, that is, more differences were found using N REisot than N REdiff (Table 3). A comparison of the difference and 15N methods performed on combined data for the 1998 and 1999 experiments showed that RE values estimated by the isotope method (mean = 51%) were significantly greater (p 
0.1) than those obtained using the difference method (mean = 47%). There was good correlation between the two methods (r = 0.77; p 0.001). Interactions involving estimation method were not significant (p > 0.1), indicating that the relationship between the methods did not change with N rate or experiment. The higher RE values obtained with the isotope method in the present study cannot be easily explained. However, these results suggest the absence of added N interactions, which are used to explain the higher RE values often reported for the difference method (Bronson et al., 2000).
Residual Soil Nitrogen
Total mineral (NH+4 NO3) N remaining in the top-60-cm soil depth at harvest was significantly higher (p < 0.1) with PCU than urea or the posthill treatment at both N rates in the standard irrigation experiment in 1999 and at the 140 kg N ha-1 rate in the excessive irrigation experiment the same year (Table 5). Similar results were reported in corn by Cartagena et al. (1995), who obtained higher residual mineral N from plots treated with CRFs compared with soluble N fertilizers. These results suggest the need for a cover crop following potato harvest if PCU is used.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Results from this study suggest that PCU may be a better option than urea in areas where N leaching from potato fields is a serious problem. However, the additional cost associated with the use of PCU to reduce leaching cannot currently be justified (Zvomuya and Rosen, 2001) without economic values being placed on the quality of the ground water resource.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSNOTESRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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