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a INIA La Estanzuela, CC 39173 Colonia, Uruguay
b 279 Plant Science, Univ. of Nebraska, Lincoln, NE 68583-0915
c Dep. of Biosystems Engineering, Univ. of Nebraska, Lincoln, NE 68583-0726
* Corresponding author (cwortmann2{at}unl.edu)
Received for publication November 10, 2006.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: ARDC, Agricultural Research & Development Center • Ch20, chisel tillage with 10-cm wide twisted shanks at 20-cm depth • Ch30, chisel tillage with 10-cm wide twisted shanks at 30-cm depth • cPOC, coarse particulate organic C (2000–250 µm) • disk, tandem disk tillage • fPOC, fine particulate organic C (250–53 µm) • miniMP, mini-moldboard plow • MP, moldboard plow • NT, continuous no-till • oxidC, permanganate-oxidizable C (0.02 M KMnO4 solution) • POC, particulate organic C • RMF, Rogers Memorial Farm • SOC, soil organic C • SOM, soil organic matter • totPOC, total particulate organic C (2000–53 µm)
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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There may be increased C sequestration with NT relative to tilled systems, at least during the first several years following conversion (Six et al., 2000), but long-term C sequestration in NT soils often does not occur (Paustian et al., 1997). In 25-yr-old tillage experiments, the most rapid changes in soil organic C (SOC) levels under NT, relative to conventional tillage, occurred during the first 10 yr (Dick et al., 1991; Paustian et al., 1997; Omonode et al., 2006). The gains in SOC in NT compared with conventional tillage are greatest near the surface, but are often negative at lower depths (Doran, 1987; West and Post, 2002). Six et al. (2002) reviewed and summarized a number of studies on SOC dynamics and stabilization, and found no indication of indefinite SOC accumulation under NT.
Occasional tillage of NT may refer to the practice of one-time tillage, conducted once in 10 or more years, in an otherwise continuous NT system. Pierce et al. (1994) found that the amount of SOC in a loam soil in Michigan that had been under NT for 7 yr was reduced for the 0- to 5-cm depth but increased at the 5- to 10-cm and 10- to 15-cm depths for 5 yr after one-time plow tillage. In semiarid western Nebraska, Kettler et al. (2000) found that at 5 yr after tillage the amount of SOC was 20% less in the 0- to 7.5-cm depth compared with undisturbed NT, but 15% more in the 7.5- to 15-cm depth. Stockfisch et al. (1999) observed a 14% decrease in SOC mass in the 0- to 50-cm depth after MP tillage of a silt loam soil in Germany that had been under minimum tillage for 20 yr. They concluded that SOC gained as a result of long-term minimum tillage was completely lost due to a one-time inversion tillage operation. Soil organic C mass was determined in this study using soil bulk density for individual soil layers measured before plowing; this is expected to result in a lower estimate of SOC mass than if the equivalent mass basis were used. On the other hand, VandenBygaart and Kay (2004) did not find a change in SOC quantity on an equivalent mass basis, except for one sandy loam soil with low SOC, at 18 mo after a one-time plowing of a field with 22 yr of continuous NT in southern Ontario, Canada. Most of the SOC gained under NT for the sandy loam was in the occluded particulate fraction that was more susceptible to tillage-induced mineralization than humified SOC, which dominated the SOC gains for the fine-texture soils (Yang and Kay, 2001).
An immediate increase in CO2 flux from soil is expected following tillage as CO2 is released with the break-up of soil aggregates and, later, with increased microbial activity due to greater access to labile SOC and increased aeration. Rates of decomposition of SOM components vary widely and continuously from easily decomposed to very stable components. Qualitative pools have been defined to facilitate interpretation. Labile SOC is considered to be more easily decomposed by soil microbes and lost due to tillage than humic SOC (Woomer et al., 1994; Grandy et al., 2006). Two labile organic C pools are commonly defined, with different decomposition rates and turnover times (Parton et al., 1987). The active C pool is composed mainly of microbial biomass, soluble carbohydrates, and exocellular enzymes, and may be much affected by tillage in the short term (Weil et al., 2003). The active C pool may be represented as permanganate oxidizable SOC. The second, slower functional pool of labile SOC is particulate organic matter (POC), defined as between 53 and 2000 µm in size. Microbial decomposition of POC may not be evident shortly after tillage, but changes in land use and soil management are expected to affect POM more than total SOC (Gajda et al., 2001).
As part of a study to determine effects of occasional tillage on NT systems (García et al., 2007; Quincke et al., 2007), this research addressed the hypothesis that deep inversion tillage could be done with minimal short-term loss of SOC. The objective was to determine the effect of different one-time tillage operations on short-term CO2 losses, and on the mass and vertical distribution of total and labile SOC.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Rogers Memorial Farm (RMF) of the University of Nebraska–Lincoln (UN–L) is approximately 16 km east of Lincoln, NE (40°50'44'' N, 96°28'18'' W, 380 m altitude) with a Sharpsburg silty clay loam (fine, smectitic, mesic Typic Argiudolls). Conversion to NT occurred in 1991 following years of minimum tillage. The NT rotation was predominantly corn or sorghum rotated with soybean. Controlled traffic was practiced to limit soil compaction. Lime was broadcast-applied without incorporation in the fall of 1997, 1999, and 2001 at a rate of 4.5 Mg ha–1 to amend soil pH. The research at this site was conducted under a rotation of grain sorghum and soybean.
The Agricultural Research and Development Center (ARDC) is near Mead, NE, about 48 km north of Lincoln (41°10'48'' N lat, 96°28'40'' W long, 358 m altitude). The soil was a Yutan silty clay loam (fine-silty, mixed, superactive, mesic Mollic Hapludalfs). The site was under a corn–soybean rotation and converted completely to NT in 1996, with infrequent shallow tillage during the preceding 9 yr. The research at this site was conducted under a rotation of corn and soybean.
Experimental Design and Treatments
The experimental design at both sites was a randomized complete block design with split plots and four replicates. Tillage was a main plot factor at both sites, and time of tillage was another main plot factor at RMF. Subplot treatments were composted feedlot manure applied before tillage at 17.7, 27.7, and 27.6 Mg ha–1 of compost for the RMF spring and fall tillage, and the ARDC fall tillage, respectively.
The tillage treatments at RMF were: (i) moldboard plowing to 20-cm depth (MP); (ii) chisel plowing with 10-cm wide twisted shanks at either 20- or 30-cm depth (Ch20 or Ch30); (iii) disk; and (iv) continuous NT as the control (Table 1). The second main plot factor at RMF was spring (26 March) vs. fall (24 October) tillage in 2003. The respective bare soil temperatures at 10 cm depth were 1.9 and 7.6°C. The MP-plots for spring tillage were disked on 23 April to reduce surface roughness; no other secondary tillage was conducted. The NT treatment had a compost subplot treatment for the spring, but not for the fall tillage. Main plots were 24 m long and 4.6 m wide.
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Measurement of Tillage-Induced Carbon Dioxide Flux
A portable gas exchange system (LI-6200 Portable Photosynthesis System, LiCor, Lincoln NE) was used to measure the CO2 concentration of air passing through an analyzer by comparing the amount of infrared radiation absorbed to that absorbed by a reference cell of known CO2 concentration. The instrument was connected to a 995-cm3 chamber with a 78.5-cm2 circular cross-section. The beveled bottom rim of the chamber was pressed 0.5 cm into the soil during measurements. After placing the chamber on the soil, the CO2 concentration within the system was drawn below the ambient level by passing the air coming from the chamber through a soda lime scrub. Soil surface CO2 flux was determined by calculating the slope of the CO2 concentration vs. time curve over three 15-s intervals when the CO2 concentration of the system was near ambient. Within each plot the first set of flux measurements was completed at three points within 5 min after tillage. The points of measurement were marked with flags so that subsequent measurements could be repeated at the same points within each plot. In general, 3 to 5 cycles of measurements were made on the day of tillage, after which one cycle was done every 2 to 7 d until planting or until the ground was covered by snow.
Measurement of Total, Particulate, and Oxidizable Soil Organic Carbon Shortly after Tillage
Soil samples, consisting of 12 cores of 1.8-cm diameter, were taken from all treatments before or shortly after planting the first crop after tillage at depths of 0 to 2.5, 2.5 to 5, 5 to 10, 10 to 20, and 20 to 30 cm. Total SOM was determined for ground soil samples (<2 mm) by weight-loss on ignition (Nelson and Sommers, 1996). All SOM measurements were converted to estimates of SOC concentration by multiplying by the Van Bemmelen factor of 0.58.
Particulate Organic Carbon
Determination of coarse (250–2000 µm; cPOC) and fine (53–250 µm; fPOC) POC was assessed by weight-loss on ignition (450°C) according to Cambardella et al. (2001). Samples of 30-g air-dried soil were dispersed with 90 mL of 5 g L–1 sodium hexametaphosphate solution in 140-mL containers and shaken on a reciprocal shaker overnight at about 140 rpm. The dispersed soil was passed through 2000, 250, and 53 µm sieves by rinsing with tap water sequentially from the larger to smaller mesh sieves until rinsate was clear. Plant fragments (>2000 µm) and material that passed through the 53-µm sieve were discarded. The material retained in the 250- and 53-µm sieves was back-washed into pre-weighed small aluminum pans, dried at 55°C for at least 24 h, and weighed to 10–4 g to determine cPOC and fPOC, respectively. Samples were then placed in a muffle furnace and heated for 4 h after the oven temperature reached 450°C. After cooling in a desiccator containing dry silica gel, the ignited sample was weighed to 10–4 g. The loss of mass on ignition represents the mass of POC for the corresponding fraction. Soil POC concentration was calculated.
Permanganate-Oxidizable Soil Organic Carbon (OxidC)
OxidC was determined according to Weil et al. (2003) by shaking 5 g of air-dried soil at about 100 cycles min–1 for 2 min in 20 mL of a solution containing 0.02 M KMnO4 and 0.1 M CaCl2. The CaCl2 in the solution caused the soil to flocculate, leaving a clear supernatant solution after 5 to 10 min of settling. Absorbance at 550 nm was recorded after diluting 1 mL of the solution with 100 mL of water. The bleaching of the purple KMnO4 color (reduction in absorbance) was proportional to the oxidizable SOC concentration with lower absorbance readings indicating more oxidizable SOC. The amount of C oxidized was estimated assuming that 1 mol Mn7+ was reduced to Mn2+ in the oxidation of 0.75 mol (9.0 g) of C (Blair et al., 1995).
Soil Bulk Density
Soil bulk density was assessed at the same depths as for SOC pools. Two blocks at each site were sampled during the summer of 2004 for the noncompost subplots of fall-tillage treatments by taking three soil cores per plot. The fall-tillage bulk density data were also used to determine SOC pools for the spring-tillage treatments at RMF. Samples for bulk density were from the inter-row, systematically sampling both trafficked and nontrafficked areas.
Bulk density for the 0- to 2.5-cm depth was determined using a metal cylinder (15-cm inner diam. and 2.5-cm height) pressed into the soil until the upper rim was even with the soil surface. The soil was removed and placed in labeled paper bags. The metal cylinder was then lined with a fine plastic sheet and filled with sand to determine the volume of soil removed. This procedure was repeated for the 2.5- to 5-cm increment.
A soil probe with a plastic tube liner was used to determine bulk density for the 5- to 10-cm, 10- to 20-cm, and 20- to 30-cm depths. In the laboratory, soil cores were slid out of the tube and cut into segments according to the corresponding increments. The mass of the soil from all five depths was obtained after oven-drying at 105°C for 48 h.
Measurement of Soil Organic Carbon at 24 to 32 Months after Tillage
Soil samples were collected in Nov. 2005 from depths of 0 to 5, 5 to 20, and 20 to 30 cm that consisted of eight cores of 1.8-cm diameter. Four additional cores of 3.2-cm diameter were taken for the 0- to 5-cm depth. Soil samples for bulk density determination were also collected at this time according to the procedures used for the earlier bulk density determination. Only the no-compost subplots of the first three replications at each site were sampled. After grinding the entire air-dried sample with a soil grinder, a subsample of 6 to 10 g was finely ground in square glass bottles for 6 h in a roller-mill (Arnold and Schepers, 2004). About 4 g of the finely ground sample was stored in 6-mL screw-capped glass vials. Total C was analytically determined with an automatic dry combustion C analyzer that was interfaced with a continuous-flow mass spectrometer (Schepers et al., 1989). Between 25 and 26 mg of the finely ground sample was weighed to 10–3 mg accuracy and wrapped in tin capsules. Inorganic C in samples from RMF, where lime had previously been surface-applied, was accounted for using an equation determined for this field by dissolving, with HCl acid, the CaCO in five oven-dried samples of varying soil pH and determining weight loss: % inorganic C = 1.4078 x pH – 9.8918, r2 = 0.999.
Calculations
The cumulative CO2 loss was calculated on a horizontal land area basis for 5 min, 4 h, 6 d, and 30 d following tillage using numerical integration (trapezoid rule), with linear interpolation between the measured fluxes over the time interval (Reicosky, 1997).
The masses of SOC, totPOC, fPOC, cPOC, and oxidC per unit land area were determined 1 to 6 mo after tillage by soil depth in consideration of soil bulk density using two methods. The fixed-depth method uses the concentration of the element of interest, bulk density, and the thickness of each sampling layer:
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i), and thickness (ti). Soil depth D was established at 20 cm and required four soil depths with ti values of 2.5, 2.5, 5, and 10 cm thick for the first to the fourth depth. The product (ti) represented the mass of soil per unit area [(Mg m–3) x m x (103 kg Mg–1) = kg soil m–2] for each sampling depth, and is multiplied by conci (g kg–1) to give g m–2 of the SOC fraction.
Treatments with greater bulk density contain more soil mass per unit area within a given sampling depth. As a result, stock estimations on a fixed-depth basis are biased in favor of those conditions that increase soil bulk density (Ellert and Bettany, 1995; VandenBygaart and Kay, 2004). The equivalent soil-mass method corrects for this bias by interpolating a calculated stock to an established dry soil mass per unit area (kg dry soil m–2). Calculations for the equivalent soil-mass method followed Gifford and Roderick (2003). To maintain consistency of abbreviations, their equation is rewritten as follows:
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ti) for the 3 or 4 sampling depths. A dry soil mass of 250 kg m–2 corresponds to approximately the 20-cm depth in the undisturbed soil, and is the reference soil mass used in the calculations. Similar calculations were used to estimate the equivalent soil mass of SOC m–2 determined approximately 24 to 32 mo after tillage. Dry soil masses of 60, 250, and 400 kg soil m–2 were used for the 5-, 20-, and 30-cm depths, respectively. Total SOC mass was also determined according to the fixed depths method.
Statistical Analyses
The analyses of variance (ANOVA) for CO2 loss and soil C variables determined 1 to 6 mo after tillage were determined using the mixed model procedure in SAS (SAS Institute, 1989) appropriate for a randomized complete block split plot design with four replications. Separate ANOVAs were run for each sampling depth and site, treating replications as random effects, while tillage and compost treatments were fixed effects. When treatment effects were significant, means were separated using the LSD option (P = 0.05).
The ANOVAs for SOC concentration and stock at 24 to 32 mo after tillage were determined using the mixed model procedure appropriate for a randomized complete block design with three replications. The tillage time x tillage interaction at RMF was tested using a data subset that excluded NT. Because the treatments differed between sites, separate ANOVAs were run by site to test treatment effects. The tillage treatment x site interaction was tested in an ANOVA combined across sites, but excluded miniMP and Ch20, which were not used at both sites. Replications were always treated as random effects, while tillage treatments were fixed effects. Means were separated by the ANOVA-protected LSD(0.05) option.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Cumulative losses at 6 d after tillage were not affected by tillage except for increased loss with spring Ch20 at RMF (Table 2). At RMF, cumulative losses at 30 d after tillage were greater for fall Ch20, Ch30, and disk than for NT, but fall MP was not significantly different. Only spring Ch20 was significantly different than NT at RMF.
Redistribution of Soil Organic Matter
The interaction effects of tillage with compost application were not often significant for SOC fractions (Table 3). The effects of compost application were generally not significant except to increase cPOC in the 0- to 2.5-cm depth for all sites/seasons and to increase total POC at ARDC. Therefore, only the main effects of tillage treatments are presented for concentrations of total and labile SOC fractions (Table 4).
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Total POC concentration was less with MP than with NT in the 0- to 2.5-cm layer in all three sites/tillage times, similar at the 2.5- to 5-cm layer, and occasionally more at the 5- to 10-cm and 10- to 20-cm depths (Table 3 and 4). Total POC was less with miniMP tillage compared with NT at 0- to 2.5-cm, but more than MP for the 0- to 5-cm layers. Total POC was more with chisel plowing than MP but generally less than NT for the 0- to 5-cm layer. Total POC concentration was greater with Ch20 and Ch30 than NT and MP at the 2.5- to 5-cm depth, but was similar for Ch20, Ch30, miniMP, and NT and occasionally more with MP at the 5- to 30-cm depth. Total POC with disking was similar to NT at all depths.
Fine POC concentration was less for MP than NT in the surface layer in all three sites/tillage times, but the tillage effect at the 2.5- to 5-cm depth was significant only at RMF with spring tillage (Table 3 and 4). Concentration of fPOC was occasionally more with MP than NT for the 5- to 20-cm depths. MiniMP, Ch20, Ch30, and disk did not affect fPOC compared with NT except for a reduction with Ch20 and Ch30 at RMF with spring tillage at the 0- to 2.5-cm depth and an increase with Ch30 at 5- to 10-cm at RMF with fall tillage.
Coarse POC concentration was lower for MP than NT in the surface layer, but not in the 2.5- to 5-cm layer (Table 3 and 4). Between the 5- and 20-cm depth, MP generally increased cPOC relative to NT, but had no effect at the 20- to 30-cm layer. MiniMP was not different from NT and MP in the surface layer. Ch20 and Ch30 did not reduce cPOC at 0- to 2.5-cm compared with NT, increased it in the 2.5- to 5-cm layer, and had no effect for other soil depths. Disking did not affect cPOC. Concentration of cPOC was increased with MP and miniMP incorporation of compost in the 10- to 20-cm and the 0- to 5-cm depths, respectively.
Concentration of oxidC was consistently lower for MP than NT in the surface layer, and in the 2.5- to 5-cm layer with spring tillage at RMF (Tables 3 and 4). Increases in oxidC with MP compared with NT were often significant at the 5- and 10-cm depth, but not at the 10- to 30-cm depths. MiniMP reduced oxidC at the surface layer compared with NT, but increased it at the 2.5- to 5-cm layer. OxidC in the 2.5- to 5-cm layer with miniMP was higher than with MP. In the 10- to 20-cm layer, miniMP had lower oxidC than MP. Chisel plowing reduced oxidC in the surface layer relative to NT but increased oxidC in the 5- to 10-cm layer. Disking did not affect oxidC compared with NT in any layer.
Total and Labile Soil Organic Carbon Stocks Shortly after Tillage
The mass of SOC stock was calculated from SOC concentration and bulk density (Table 4 and 5). Bulk density was generally less following one-time tillage than with NT.
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Soil Organic Carbon at 24 to 32 Months after Tillage
The bulk density values used in estimation of SOC stocks at this time are presented in Table 7. The effect of the season by tillage interaction was not significant for SOC stock at RMF (Table 8) and data from fall and spring tillage at RMF were combined to determine tillage effects on SOC.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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These results contrast with the findings of Reicosky and Lindstrom (1993) and of Reicosky (1997) where CO2 loss was five times as much during the 19 d following MP tillage in early September compared with continuous NT, while losses with other tillage operations were intermediate. The cumulative CO2 losses during the first 5 h after tillage were much higher than in the current study. In the earlier studies, CO2 loss was greater with the deeper tillage and greater soil disturbance associated with MP tillage than with other implements. This contrasts with the current findings where cumulative losses were not greater with MP than with NT. The differing results may be due to soil temperature at the time of tillage. Tillage in the current study was conducted when soil temperatures at the 10-cm depth were between 1 to <8°C.
Due to low soil temperature at the time of tillage in the current study, an immediate flush of CO2 with tillage due to physical release of soil air was expected to be small. Earlier studies ascribed the CO2 losses measured immediately after tillage to increased microbial activity (Reicosky and Lindstrom, 1993) as microbial decomposition of SOM is increased with aggregate disruption (Rovira and Greacen, 1957). Other studies found that the immediate flush of CO2 with tillage was mostly due to physical release of soil air. Concentration of CO2 in soil air may be 5 to 12 x104 µL L–1 during the summer compared to 2 x104 µL L–1 in the fall or early spring. The importance of physical release was shown in a study where repeating tillage several times did not substantially increase CO2 losses (Ellert and Janzen, 1999), and by Wuest et al. (2003), who observed a CO2 flush resulting from disturbance of previously sterilized soil to be comparable to the flush from disturbance of fresh soil.
The type of chamber used in measurement of CO2 flux may be important. A flush of CO2 flux immediately after soil disturbance has been observed when using a soil chamber of similar type and size as in this study (Prior et al., 1997, 2004; Rochette and Angers, 1999) and with a larger canopy chamber (Reicosky and Lindstrom, 1993; Reicosky et al., 2005). Reicosky and Lindstrom (1993) and Reicosky et al. (1997) commonly found greater CO2 flushes measured with canopy chambers than with smaller soil chambers as used in this study. This is probably due to more soil roughness under the canopy chamber (Reicosky et al., 1997) and increased turbulence and dynamic pressure inside the canopy chamber produced by the mixing fans (Hanson et al., 1993; Dugas et al., 1997). Reicosky et al. (1997) found that measured CO2 fluxes with the two chamber types were similar for undisturbed soil, but measurements for tilled soil were greater with a larger chamber than with the smaller soil chamber.
Several factors may have affected the accuracy of CO2 fluxes with the soil chamber.
Redistribution of Soil Organic Matter within the Profile
Chisel, mini-MP, and MP tillage often resulted in reduced SOC in the surface depth of 2.5 cm with the greatest reduction due to MP tillage. Increases in SOC at depths below 5 cm were generally not significant for total SOC but more frequent for POC. Tillage can cause vertical redistribution of SOC (Angers et al., 1997; Lorenz and Lal, 2005; Grandy and Robertson, 2006). Chisel tillage disturbed soil to 20- or 30-cm depth, but with little inversion, and rarely reduced SOC fractions in the 0- to 2.5-cm layer, but an increase in the 2.5- to 5-cm layer was often observed. Disk tillage had little effect on SOC stratification. The effectiveness of tillage implements for reducing SOC stratification in long-term NT systems appears to be: MP > miniMP > Ch30 = Ch20 > Disk = NT.
Soil Organic Carbon Stocks Shortly after Tillage
The more traditional "fixed-depth" and "equivalent soil mass" methods gave contrasting results, as expected. Given the effect of tillage on soil bulk density, the soil mass per unit area to a fixed depth of 20 cm was consistently lower for tilled than for NT soils (Table 5 and 7). The "fixed depth" approach to the 20-cm depth indicated SOC loss with some tillage operations, but less difference in SOC stocks between NT and tilled soils was found with the equivalent mass method. Similar findings were reported by Ellert and Bettany (1995) after reassessing previously published data on C storage for NT vs. plowed soils.
Soil Organic Carbon Stocks at 24 to 32 Months after Tillage
The SOC mass in 400 kg m–2 of soil at 24 to 32 mo after tillage was similar for NT and MP tillage (Fig. 2), while SOC at the 0- to 5-cm depth was still considerably less with MP than with NT (Fig. 1). The results indicated that tillage to invert surface soil, which is relatively high in labile SOC, can be done without increased SOC loss while leaving a surface soil relatively low in SOC and presumably with increased potential for C sequestration compared with continuous NT. Assuming that a greater proportion of the inverted SOC will be converted to humus rather than lost during decomposition compared with NT, this deeper SOC will not easily be lost if one-time tillage is practiced again, for example, 10 or more years in the future.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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REFERENCES |
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