Geoderma 156 (2010) 207215

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Geoderma
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e o d e r m a

Tillage and phosphorus management effects on enzyme-labile bioactive phosphorus availability in Cerrado Oxisols
P.S. Pavinato b,1, T.H. Dao a,, C.A. Rosolem b
a b

US Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA Dept. of Crop Science, Sao Paulo State Univ. (UNESP/FCA), Botucatu, Sao Paulo, Brazil

a r t i c l e

i n f o

a b s t r a c t
Phosphorus (P) is an essential element in crop nutrition, which can be growth limiting or an environmental contaminant, if present in excess. Tillage practices have a direct effect on the behavior and availability of soil P. Sorption and availability of various P forms were evaluated in an incubationfractionation study of three soils, a Typic Paleudults (CR soil) and two Cerrado Oxisols (Latossolo VermelhoAmarelo [LVA] and Latossolo Vermelho [LV]) with distinct biogeochemical characteristics and tillage management history. Phosphate and myo-inositol hexakisphosphate (mIPH) were strongly sorbed by the soils. Maximum adsorption capacities (Smax) were 2.26.9, 3.37.8, and 1.619.8 mmol kg 1 for phosphate in the 040 cm depths of the CR, LV, and LVA soils, respectively. For mIPH, Smax were 1.23.7, 3.75.5, and 4.65.2 mmol kg 1. Saturation indices reected the long-term effect of repeated manure applications on the Paleudults and the near saturation of its P holding capacity, in contrast to the recently cultivated Cerrado soils. Tillage method appeared to have altered P retention characteristics of the near-surface zone very slightly, while increases in ligandexchangeable (EEPi) and enzyme-labile organic P (EDTA-PHP) forms were observed in no-till Oxisols. In the Paleudults, added manure P increased bioactive P fractions and P saturation of no-till near-surface soil zone. Estimates of all bioactive P fractions using the ligand-based enzymatic assay showed it to be an effective method for assessing P availability in soil and developing sustainable P management strategies, particularly in Cerrado Oxisols that were low in organic matter while having an extensive P-xing capacity. Published by Elsevier B.V.

Article history: Received 28 September 2009 Received in revised form 26 December 2009 Accepted 15 February 2010 Available online 11 March 2010 Keywords: Enzyme-hydrolyzable phosphorus Phytate Conventional tillage No-tillage Phosphorus sorptiondesorption

1. Introduction In natural ecosystems and P-depleted cultivated agrosystems, recycling of P has to occur through the turnover of biological tissues and the mineralization of organic P forms in order to meet plant nutritional requirements. Phosphomonoesters are the most stable and predominant forms of organic P in soils, including myo-inositol hexakisphosphoric acid, i.e., phytic acid (Magid et al., 1996). myo-Inositol hexakisphosphate and lower phosphomonoesters of inositol (i.e., inositol phosphomonoesters) may constitute up to 50% of total soil organic P (Wild and Oke, 1966; Koopmans et al., 2003; Dao, 2004). Over 90% of organic P that accumulated in soils following continuous application of superphosphate fertilizers for over three decades were inositol phosphomonoesters (Condron et al., 1985). Other major forms of organic P included phosphodiesters, i.e., nucleic acids, and phospholipids (Novais and Smyth, 1999). Inositol phosphomonoesters, whether plant- or microbially-derived or added as manure P, are relatively resistant to enzymatic hydrolysis, leading to their preferential accumulation in soils
Corresponding author. Tel.: + 1 301 504 8315; fax: + 1 301 504 8162. E-mail address: thanh.dao@ars.usda.gov (T.H. Dao). 1 Present address: Federal Technological Univ. Parana (UTFPR), campus Dois Vizinhos, PR Estrada para Boa Esperana, Brazil. 0016-7061/$ see front matter. Published by Elsevier B.V. doi:10.1016/j.geoderma.2010.02.019

(Anderson et al., 1974; Stewart and Tiessen, 1987; Dao, 2004). These P forms often are complexed with polyvalent cations, and exist as sorbed or insoluble precipitates. The adsorption of myo-inositol phosphomonoesters on hydrous oxides has been described essentially by a model previously developed to describe phosphate adsorption onto goethite (Ognalaga et al., 1994; Celi et al., 2001). In a binuclear surface complex arrangement, the quantity of adsorbed phosphate-P per unit surface area averaged 2.5 mol m 2 for a number of samples of natural and synthetic goethite (Golberg and Sposito, 1984; Torrent et al., 1990). However, the formation of monodentate complexes can also account for the adsorption of phosphomonoesters onto colloids and sediments in aquatic ecosystems, forming monodentate bridges with FeO+ and AlO+ groups of more than one surface of colloids (Golberg and Sposito, 1985). Although the Fe and Al hydroxides have a high afnity for phosphate and phosphomonoesters, an increase in the desorption of these P forms is observed when polydentate ligands or organic complexing agents are present in the soil solution. These types of compounds can maintain more P in free forms in solution upon forming complexes with Al3+, Fe3+, Mn2+ and Ca2+, which would have reacted with free phosphate species to form insoluble complexed phosphates or phosphomonoesters (Lindsay, 1979; Celi et al., 2001; Dao, 2003, 2004, 2007). High organic matter content in surface soil layers has been implicated in reduced P adsorption capacity,

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which can be lower than those of subsurface layers, but allowing a greater P turnover from phosphate fertilization (Schwertmann and Taylor, 1989). Organic acids and other anions from decomposing plant and animal residues complex free anionic adsorption sites in soil, buffering these sites and preventing the adsorption of phosphate (Ohno and Crannell, 1996; Fiorucci et al., 2002). Drawing on the ligand-exchange phenomenon, Dao (2003, 2004) developed a ligand-based fractionation procedure to categorize soil and manure P species and characterize their potential hydrolysis and bioavailability. Dilute solutions of large polydentate organic anions facilitate the release of phosphate and phosphomonoesters via a ligand-exchange process. The uncomplexed P forms are subsequently readily hydrolyzed by the extracellular phosphohydrolase enzymes to yield inorganic phosphate. Organic anions occurring naturally in soils compete with phosphate for adsorption sites, but signicant differences exist in their ability to release phosphate; for example, oxalate was more efcient than formate (Fox et al., 1990). However, oxalate was much less efcient than large polycarboxylate ligands in their ability to effect organic P solubilization and hydrolysis by phosphohydrolases (Dao, 2004). Given the progressing nature of the in situ extractionhydrolysis of the procedure, a scale of extractability and bioavailability of the P pools to microbial and plant root systems was proposed (Dao, 2007). As food and feed production try to keep pace with human and livestock needs, crop production is being extended into more fragile/ marginal areas of the world. Brazilian Cerrado soils developed under a savanna type of vegetation are being cultivated although they are characteristically low in native fertility (Medina, 1993; Oberson et al., 1999). Native Cerrado soils are low in pH and have high exchangeable Al3+ and Mn2+. They are low in macro- and micronutrients and thus, chemical or organic nutrients are needed to make these soils suitable for crop production. Phosphorus is one of the nutrients that must be added at high rates. The soils are highly weathered and have high clay content, which contain a large amount of Fe and Al oxides and hydroxides; consequently, Cerrado soils have a substantial P adsorption, or sorption when exact adsorption mechanisms are not precisely characterized or known, and a great part of the applied fertilizer P is strongly sorbed and becomes unavailable to crop plants. Although the quantity of added inputs can be managed to meet crop nutritional needs, land management practices directly affect the behavior and plant availability of the applied P. In no-till production systems, fertilizer P, crop residues, and residue-derived organic matter accumulate at the surface and in the near-surface zone (Dao, 1998, 2004; Allmaras et al., 1999). No-tillage changes P availability in soil, mainly because of residue-P accumulation in the near-surface soil zone, and as crop residues decompose, they release the P contained within. In addition, organic acids produced during crop residue decomposition may promote a competition for sorption sites with P in soil, and its availability to plants and microorganisms (Rheinheimer, 2000; Fiorucci et al., 2002). Conversely, it has also been suggested that low molecular weight organic acids produced during organic matter decomposition were too low in concentrations and too labile to have any signicant effect on P sorption (Guppy et al., 2005). In a conventional production system, tillage incorporates and redistri-

butes the organic residues and residue-borne nutrients throughout the tilled soil layer. The intimate soil-residues contact enhances rapid microbial decomposition of the organic matter and the release and uniform dispersion of residue-borne P, compared to the stratication and lack of mixing in the near-surface zone in a NT soil (Dao, 1998, 2004). As limited information is available on the interactions of tillage method and P management on soil sorption and bioavailability of organic P pools, the objectives of this study were to (i) determine phosphate and organic mIPH maximum adsorption capacity of two Brazilian Cerrado Oxisols and a Typic Paleudults from Maryland, USA, and (ii) evaluate the effects of soil management practices on the soils' sorption capacity for these P species and their effects on the dispersion of bioactive fractions of P with soil depth. Inherent to the latter objective, the goal was to evaluate the possible use of the enzyme-labile fraction as a fertilization indicator for developing sustainable nutrient management practices for Cerrado Oxisols in Brazil. 2. Material and methods 2.1. Soils The soils from Brazil include a Latossolo VermelhoAmarelo (soil LVA) and Latossolo Vermelho (soil LV), according to the Brazilian soil classication system (EMBRAPA, CNPS, 1999) and both soils are Typic Hapludox, according to USDA Soil Taxonomy (USDA, NRCS, 1999), both having more than 50% of clay content in the upper 40 cm soil depth. In 2006, soil cores (040 cm) of the LVA soil were collected from elds cultivated to soybeanmaize under conventional (CT) and no-tillage (NT) management since 1992 in Luzinia, Gois (16 15 10 S, 47 56 19 W). Similarly, cores of the LV soil were collected in areas under CT and NT management since 1994, in Costa Rica, Mato Grosso do Sul (18 12 40 S, 53 12 13 W). The elds were cropped to a rotation of maizeradishcottonsoybean. Samples of a Christiana silt loam (soil CR) were also included in this study. They were collected from a long-term study of managing high-P soils and developing management practices to reduce P losses from these soils on the USDA-ARS Beltsville Agricultural Research Center's farm (39.03 N and 76.85 W). The Christiana soil is classied as ne, kaolinitic, mesic Typic Paleudults (USDA, NRCS, 1999). In May 2007, soil cores (040 cm) were collected from NT plots planted to orchardgrassred clover amended with two rates (0 and 30 mg manure P kg 1) of dairy manure. Soil cores were obtained from the 0 40 cm depth. Samples were dried, ground to pass a 2-mm sieve. All soil samples were stored dry prior to conducting P fractionation and analysis. Soil classication and selected properties of the soils are presented in Table 1. 2.2. Phosphate and myo-inositol hexakisphosphate sorption and desorption Phosphate adsorption capacity of the soils was determined by shaking a mixture of samples and aliquots of KH2PO4 solutions (0, 0.25, 0.75, 1.5, 3.5, 5, and 6.5 mmol L 1 of P). Each sorption batch

Table 1 Selected properties of the two Brazilian Cerrado Oxisols and a temperate zone Typic Paleudults under no-till (NT) and conventional (CT) management. Soil name Classication pH CaCl2
1

CEC mmol kg 1

Organic C g kg 1 26 28 22

Total P mg kg 1 449 604 804

Mehlich 3-P mg kg 1 12.2 17.6 65.7

2

Exchangeable Al3+ mmol kg 1 0.1 0.4 n.d.3

Oxalate-extractable Fe3+ g kg 1 1.9 2.7 n.d.

LVA LV Christiana
1 2 3

Latossolo VermelhoAmarelo Latossolo Vermelho Fine, kaolinitic, mesic Typic Paleudults

5.73 5.95 5.2

87 116 173

In 1:2 soil to 0.01 M CaC l2. Mehlich 3-extractable P (Mehlich, 1984). n.d. = not determined.

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consisted of duplicates of 1-g samples (oven-dry equivalent) of airdry soil added to 10 ml of one of the equilibrating solutions in plastic centrifuge tubes (160 8 mm). After 18 h at 150 rotations per minute (rpm), the soil suspensions were centrifuged at 10,000 g for 10 min, and phosphate-P remaining in the solution phase was measured. The quantity of phosphate-P adsorbed was estimated by the difference between the initial and the nal P concentration in solution. myo-Inositol hexakisphosphate sorption characteristics were determined for the LVA, LV, and CR soils. Initial solution-P concentrations were slightly different for the CR soil and the LVA and LV soils, given the expected differences in sorption potential between Ultisols and Oxisols. The following mIPH concentration ranges, (0, 0.1, 1, 2, 5, and 7.5 mmol L 1) and (0, 0.5, 1, 2, 5, and 7.5 mmol L 1) were used for the CR soil, and the LVA and LV soils, respectively. Similar to phosphate-P sorption experiments, each mIPH sorption batch consisted of duplicates of 1-g sample (oven-dry equivalent) of air-dry soil added to 10 ml of one of the equilibrating solutions in 160 8 mm plastic centrifuge tubes. After shaking for 18 h and centrifugation (10,000 g for 10 min), a 3-mL aliquot of the supernatant solution was transferred to another clean test tube; an aliquot of a stock solution containing 0.5 unit mL 1 of phosphohydrolases (Dao and Hoang, 2008) was added, and the mixture was re-equilibrated for 18 h at room temperature. myo-Inositol hexakisphosphate remaining in solution after equilibration with samples of the three soils were hydrolyzed to release phosphate and their concentrations were determined colorimetrically (APHA, 1998). Following the measurement of mIPH adsorption on the LVA, LV, and CR soils, 6 mL of the equilibrium solution phase were removed and replaced with deionized water to determine the desorption of mIPH from the adsorbed state. The solid phase was re-suspended and the soilwater suspension was shaken with a gyratory shaker (150 rpm) for 18 h. Then, the suspension was centrifuged at 10,000 g for 10 min. Six mL of the solution phase were removed and replaced with deionized water. An aliquot of this supernatant was used to determine mIPH-P as previously outlined. Meanwhile, the suspension was re-equilibrated for another 18 h, and the sequence of isolation of supernatant and replacement with water, and analysis for mIPH desorbed in the solution phase was repeated for ve cycles. 2.3. Soil P fractionation Bioactive P fractions in soils were determined according to Dao (2004, 2007). The fractions included: 1) water-extractable P (WEP), 2) ethylenediamine-N,N,N,N-tetraacetate (EDTA)-exchangeable inorganic phosphate-P (EEPi), 3) the EDTA-phosphohydrolase-labile P (EDTA-PHP), and 4) the all-inclusive total bioactive P (WEP + EEPi + EDTA-PHP) which were determined as follows. The P fraction extracted by water (WEP) and a 5 mmol L 1 EDTA solution (EEPi) were measured in soilextractant mixtures (1:100, w/v) that were shaken horizontally at 150 rpm for 1 h. Samples were centrifuged by 10,000 g for 10 min; a subsample of the supernatant (0.1 mL) was used for phosphate-P analysis. In the sample used for the EEPi measurements, an aliquot (1 mL) of a stock solution of phosphohydrolase enzymes was added and the mixture was again shaken for 18 h, followed by centrifugation and collection of the supernatant for phosphate-P analysis. An all-inclusive total bioactive P fraction in soil was also determined. Phosphohydrolases were added to biologically degrade the organic P partially extracted by EDTA during the rst hour of equilibration, but not previously measured by the colorimetric method (i.e., due to the short turnaround time), and the additional organic P released during the overnight equilibration with the enzymes; details of the rationale and the fractionation protocols are found in Dao (2003, 2004) and Dao and Hoang (2008). The organic EDTA-exchangeable phosphohydrolase-labile P (EDTA-PHP) was calculated as the difference between the all-inclusive total bioactive

P and the corresponding EEPi fractions. The P fraction measured after phosphohydrolases addition called EDTA-PHP is also considered available to microorganisms and plants (Dao, 2004; Rao and Dao, 2008; Dao, in press). All extracted and hydrolyzed phosphate-P concentrations were quantied using the blue molybdateascorbic acid method on an ion-analyzer (APHA, 1998). Total elemental Al and Fe in soil were determined by X-ray uorescence spectrometry, using pulverized soil samples (Dao and Zhang, 2007). Estimates of plant-available Al, Fe, and P were obtained according to the Mehlich-3 procedure (Mehlich, 1984) to calculate a soil phosphorus saturation index (PSIM3). The soil-Mehlich-3 suspension (1:100, w/v) was mechanically shaken for 5 min, ltered, and the ltrate collected for metal and P analysis (APHA, 1998). 2.4. Statistical analysis and numerical methods The phosphate and mIPH sorption data for all three soils were tted to the Langmuir adsorption model:
Ce

.
S

Ce

.
Smax

.
Kb

where C is the equilibrium P concentration in solution (mmol L 1), S = sorbed-phase P, (mmol kg 1), Kb is the soil P afnity index (L mmol 1), and Smax is the P sorption maximum (mmol kg 1). The soil P saturation degree is dened as the quantity of P adsorbed divided by the soil adsorption capacity (Schoumans, 2000). It is used to estimate the fraction of adsorption sites effectively occupied by P and the P desorption potential. Phosphorus saturation index (PSIM3) was also calculated according to the following equation: PSIM3 = PM3 =AlM3
+ FeM3

where XM3 are Mehlich 3-extractable P, Al, or P concentrations (mmol kg 1). The P sorption and fractionation experiments were established according to a randomized complete block design. Management and soils effects on P sorptiondesorption capacities were evaluated using PROC MIXED of the Statistical Analysis System (SAS) Ver. 9.1 (SAS Institute, 2004). The goodness-of-t of the sorptiondesorption data to the Langmuir adsorption model was determined using PROC GLM. Treatment means were compared using the Student's LSD test at the 0.05 level of probability. In addition to the coefcient of determination (r2), the root mean squared error (RMSE) was also calculated to describe the goodness-of-t of the data. 3. Results and discussion 3.1. Phosphorus sorptiondesorption The high sorption capacity of the three soils for phosphate and mIPH are shown in Tables 2 and 3, respectively. Sorption of phosphate and mIPH increased with increasing P concentrations and never actually reached a saturation plateau in the two Oxisols over the P concentration ranges of 0.256.5 mmol L 1 and 0.057.5 mmol L 1, respectively; these ranges encompassed P concentrations normally encountered in crop production systems. The order of increasing phosphate sorption capacity of the soils was CR=LVb LVA (Table 2). The Smax ranged between 2.2 and 6.9 mmol kg 1 for phosphate sorption isotherms conforming to the Langmuir model with r2 N 0.78 in the CR soil. The Langmuir model failed to describe the behavior of phosphate in the 1540 cm depth of the CR soil; the curvature of the isotherm at high phosphate concentrations reected a decreasing sorption capacity and different sorption mechanisms. The LV soil showed Smax values in the same or slightly higher range as that of CR soil. The LVA showed the highest potential sorption capacity for phosphate, with Smax

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P.S. Pavinato et al. / Geoderma 156 (2010) 207215 Table 3 Effects of phosphorus and tillage management on soil retention of myo-inositol hexakisphosphate (i.e., phytic acid) as described by the Langmuir adsorption model in two Brazilian Cerrado Oxisols and a Paleudults. Soil Tillage Phosphorus Soil method management depth cm LVA Conv. tillage Conv. 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 Smax
a

Table 2 Effects of phosphorus and tillage management on soil retention of phosphate as described by the Langmuir adsorption model in two Brazilian Cerrado Oxisols and a Paleudults. Soil Tillage method Phosphorus Soil management depth cm LVA Conv. tillage Conv. 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 05 510 1015 1525 2540 Smax
a

mmol kg 1 19.8 12.3 11.7 14.0 17.0 11.6 11.6 14.5 12.1 13.6 5.0 7.0 3.3 5.5 7.3 7.8 3.4 4.1 7.4 6.2 2.2 5.2 4.7 8.6 6.9 2.5 5.3 4.2 6.4 9.0 a cd d bc ab d d b cd c b b c b b a c bc b b b ab b ab ab bc ab b ab a

Coefcient of RMSE b determination r2 0.789 0.933 0.949 0.985 0.977 0.964 0.970 0.965 0.992 0.997 0.972 0.930 0.792 0.990 0.993 0.888 0.983 0.991 0.953 0.998 0.960 0.977 0.824 0.613 0.993 0.957 0.980 0.788 0.353 0.281 0.013 0.013 0.012 0.005 0.004 0.010 0.009 0.007 0.004 0.002 0.030 0.036 0.072 0.016 0.009 0.040 0.036 0.022 0.024 0.006 0.134 0.038 0.107 0.087 0.012 0.120 0.035 0.127 0.245 0.091

mmol kg 1 5.2 a 5.1 a 4.5 b 5.0 a nd 5.3 a 4.6 b 5.0 a 5.0 ab nd 3.9 bc 4.2 b 3.7 c 5.3 a nd 4.7 b 4.3 b 5.5 a 5.4 a nd 2.7 a 1.8 b 1.7 bc 1.4 c nd 2.9 ab 3.7 a 2.3 b 1.2 c nd

Coefcient of RMSE b determination r2 0.969 0.984 0.987 0.984 0.998 0.990 0.986 0.977 0.990 0.991 0.967 0.970 0.990 0.996 0.950 0.966 0.957 0.978 0.860 0.952 0.856 0.629 0.884 0.840 0.009 0.007 0.007 0.007 0.002 0.006 0.006 0.008 0.008 0.006 0.015 0.008 0.006 0.004 0.010 0.009 0.026 0.030 0.083 0.059 0.045 0.061 0.052 0.134

Notillage

Conv.

Notillage

Conv.

LV

Conv. tillage

Conv.

LV

Conv. Tillage

Conv.

Notillage

Conv.

Notillage

Conv.

Christiana NoSiL tillage

Control

Christiana NoSiL tillage

Control

Notillage

Manure added

Notillage

Manure added

Model parameters followed by the same letter within a soil are not signicantly different according to HSD test at the 0.05 level of probability. a Langmuir adsorption model: Ce/S = Ce/Smax + 1/Kb, where 1/Smax is the slope of the plot of Ce/S (kg L 1) vs. Ce (mmol kg 1). b RMSE = root mean squared error.

Model parameters followed by the same letter within a soil are not signicantly different according to HSD test at the 0.05 level of probability. nd = not determined. a Langmuir adsorption model: Ce/S = Ce/Smax + 1/Kb, where 1/Smax is the slope of the plot of Ce/S (kg L 1) vs. Ce (mmol kg 1). b RMSE = root mean squared error.

values being 2 to 3-fold higher than those of the CR or LV soils. These results were in general agreement with the published literature (Anghinoni et al., 1996; Varinderpal et al., 2006). Varinderpal et al. (2006) reported that P adsorption capacity in Indian soils is low, when compared to Brazilian soils, showing a P sorption Smax of about 5.5 mmol kg 1 in soils that had not been fertilized for over 20 years. When the soil was fertilized at the recommended rates, Smax was reduced to 3.5 mmol kg 1. This trend in Smax between the three soils in our study can be explained by the P saturation indices of the soils (Tables 4 and 5). The PSIM3 values in the CR soil were signicantly higher than the indices for the two Oxisols, with values varying between 11 and 19% for the 0 15 cm soil depth where manure was applied. These elevated concentrations of extractable soil P reduced the availability of sites for retaining or sorbing additional P. The adsorption maxima of the soils for mIPH followed the same order for that of phosphate, although the net differences in Smax for the organic P form were much smaller between soils (Table 3). The same explanation presented for the differential sorption of phosphate between the three soils can be extended to the observed reduction in the sorption of mIPH in the CR soil, when compared to the two Oxisols (Table 5). On a molar basis, it appeared that the sorption of mIPH onto soils was less than that of phosphate; however, it must be noted that, on an elemental P basis, a greater concentration of P was sorbed in the form of mIPH than in the form of phosphate. The large molecule has been postulated to form stable monodentate and binuclear bridges with FeO+ and AlO+ of surfaces of soil colloids or sediments (Golberg and Sposito, 1985).

Solution pH affects the distribution of dissociated species of phosphate and mIPH in the solution phase and therefore the extent of their sorption onto the three soils (Dao, 2003, 2004; Sato and Comerford, 2005; Dao, 2007); however, the CR soil being the most acidic of the three actually showed the lowest Smax for both inorganic and organic P forms, at least in the 05 or the 510 cm soil depth (Tables 13). The pH effect was overshadowed by other soil properties, namely, the content of Al and Fe oxy-hydroxides. Clay and oxides

Table 4 Mehlich-3 Extractable Phosphorus in two Brazilian Cerrado Oxisols and a Paleudults as affected by tillage method and manure phosphorus amendment. Soil and P management1 Soil depth (cm) 05 510 1015 1525 2540 Mean

mg of P kg 1 LVA-CT LVA-NT LV-CT LV-NT CR-NT-CTRL CR-NT-manure added Mean LSD0.05

1

12.2 13.1 17.6 15.5 65.7 138.0 43.7 21.4

13.1 10.5 19.1 29.1 61.0 103.0 39.3 14.3

10.6 7.2 21.3 23.4 54.1 74.0 31.8 16.3

1.7 4.0 8.8 12.9 36.9 42.6 17.8 15.0

0.8 1.0 2.1 3.7 7.3 10.4 4.2 3.9

7.7 7.2 13.8 16.9 45.0 73.6 14.6

CT = conventional tillage; NT= no-tillage; CTRL = 0 kg P ha 1; Manured = 30 kg manure P ha 1. Treatment means followed by the same letter are not signicantly different according to the Student's Least Signicant Difference at 0.05 level of probability.

P.S. Pavinato et al. / Geoderma 156 (2010) 207215 Table 5 Phosphorus saturation index (PSIM3) of two Brazilian Cerrado Oxisols and a Paleudults as affected by tillage method and manure phosphorus amendment. Soil and P managementa Soil depth (cm) 05 % LVA-CT LVA-NT LV-CT LV-NT CR-NT-CTRL CR-NT-manure added Mean LSD0.05 1.4 1.8 1.9 2.1 9.8 18.6 5.9 3.1 1.5 1.1 2.3 3.1 9.6 14.4 5.3 1.9 1.2 0.7 2.1 2 8.7 10.6 4.2 2 0.2 0.4 0.8 1.1 6.7 6.6 2.6 1.9 0.1 0.1 0.2 0.3 1.2 1.6 0.6 0.4 0.9 0.8 1.5 1.7 7.2 10.4 2 510 1015 1525 2540 Mean

211

the LVA and LV soils, respectively; up to 18% was desorbed from the CR-CTRL soil. However, it has been previously observed that, rather than being protected from decomposition by being tightly sorbed on soil surfaces, mIPH, and in general, organic P, are desorbed and mineralized where dephosphorylation is assisted by organic ligands (Dao, 2004; Dao et al., 2005; Rao and Dao, 2008; Dao, in press). 3.2. Soil bioactive P fractions The relative proportions of extractable P fractions at each depth of the three soils are presented in Figs. 13. Water-extractable P (WEP) was present at concentrations b1012 mg kg 1 in the Oxisols, and the LVA soil had the lowest WEP concentrations among the three soils (Figs. 1 and 2). The results also suggested that no-till management of the LVA soil had increased the WEP fraction, either at the expense of the ligand-exchangeable or EEPi fraction, or by the solubilization mineralization of an additional refractory fraction of total P, particularly at soil depths between 5 and 40 cm. Overall, the levels are very low, therefore, the continuous replenishment of the WEP pool is critical for meeting the nutritional requirements of plants. Weakly sorbed labile P fractions are in equilibrium with the soil solution and are sequentially measured as EEPi and EDTA-PHP fractions. 3.2.1. EEPi Adding the ligand EDTA to the water extracting solution (5 mmol L 1) resulted in the release of weakly held inorganic complexed phosphates on the exchange complex of soil mineral and organic matter surfaces. The EEPi pool could be considered potentially available to plants and microbial uptake. The short-term EDTA extraction and immediate analysis for phosphate-P yielded mostly inorganic forms that were present in the extracts of the soils (Dao, 2003). The EEPi fraction was previously found to be equivalent to the Mehlich 3-P pool in soil (Dao, 2004; Dao et al., 2005) or in manure (Dao et al., 2006; Dao and Zhang, 2007). Again, this was the case for these three soils (Table 2 and Figs. 13). The EEPi fraction decreased with soil depth in the LVA and CR soils, while it remained constant in the 015 cm depth of the LV soil then

Treatment means followed by the same letter are not signicantly different according to the Student's Least Signicant Difference at 0.05 level of probability. a CT = conventional tillage; NT = no-tillage; CTRL = 0 kg P ha 1; Manured = 30 kg manure P ha 1.

content have a strong inuence on soil P adsorption capacity. Rheinheimer (2000) showed that the mean Smax was 1130 mg kg 1 in the 017.5 cm soil depth of an Oxisol similar to the LV soil, with high clay content (680 g kg 1). The Smax varied from 915 mg kg 1 in the 0 2.5 cm depth, to 1075 mg kg 1 in the 7.517.5 cm soil depth of another Oxisol that contained less clay (580 g kg 1). The effect of soil depth on P sorption differed between the two P forms; Smax for phosphate were increasing with soil depth, while those for mIPH were unchanged or slightly declining (Tables 2 and 3). The apparent divergence and array of possible adsorption mechanisms reected one of the weaknesses of relying solely on sorption isotherm data for predicting the availability of phosphate- and mIPH-P in soil. For example, the desorption results for the 05-cm depth of the three soils showed that mIPH was hard to desorb from these soils, once exposed to them (Fig. 5). After ve cycles of desorption with over 30 mL of water (i.e., an equivalent of over 100 pore volumes), relatively small quantities (i.e., between 1.5 to 5% and 0 to 8.5%) of the adsorbed mIPH were displaced and released to the solution phase of

Fig. 1. Effect of conventional (CT) and no-tillage (NT) management on water-extractable (WEP), EDTA-extractable (EEPi), and phosphohydrolase-labile phosphorus (EDTAPHP) concentrations in the 040 cm depth of the LVA soil.

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Fig. 2. Effect of conventional (CT) and no-tillage (NT) management on water-extractable (WEP), EDTA-extractable (EEPi), and phosphohydrolase-labile phosphorus (EDTAPHP) concentrations in the 040 cm depth of the LV soil.

declined to native P levels deep in the prole unaffected by tillage management, as in the other two soils. The EEPi concentrations in all three soils were higher than the WEP concentrations, representing stocks of inorganic phosphates that can be solubilized and released to the soil solution. In the 05 cm depth, the EEPi amounted to an increase of 78% more phosphate in solution, compared to that

extracted by water alone in the LVA and LV soils; meanwhile phosphate desorbed by EDTA was more than 6.2 and 6.9 times that extracted by water alone from the untreated and the manured CR soil, respectively. The EEPi fraction represented about 22% of soil total P in the 05 cm depth of the CR soil. In comparison, the EEPi fraction represented only 1.9 and 2.1% of total P in the same soil depth of the

Fig. 3. Effect of manure phosphorus amendment on water-extractable (WEP), EDTA-extractable (EEPi), and phosphohydrolase-labile phosphorus (EDTA-PHP) in the 040 cm depth of no-till Christiana soil. CTRL = control 0 kg P ha 1; manured = 30 kg manure P ha 1.

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213

NT-LVA and LV soils, respectively. These data for the CR soil are in agreement with the results reported by Dao (2004) and Green et al. (2006), where the EEPi fraction represented from 22 to 41% of soil total P. No data currently exist in the literature for additional comparisons of the LVA and LV results. The effects of tillage method on soil EEPi were observed in the LVA soil, but not the LV soil (Fig. 1); NT soil had similar concentration of EEPi in the surface 05 cm depth but then showed higher EEPi levels in the 515 cm soil depth and then declined to the same EEPi concentrations observed for the soil under CT management. We postulated that tilling the soil mixed and diluted the fertilizer phosphate with a larger volume of untreated soil, contributing to lower EEPi concentrations and their extractability in the 540 cm depth of the tilled Oxisols. 3.2.2. EDTA-PHP In combination with the EDTA ligand, the phosphohydrolase enzymes continuously extract, dissolve, and hydrolyze soil organic P in situ during the 18-h equilibration period at ambient temperature (22 C). These reactions were driven or compelled to reach an equilibrium, preferentially toward the dephosphorylation of soil organic P forms. This in situ approach is different from other extraction and enzymatic methods (e.g., Hedley et al., 1982; He and Honeycutt, 2001). In these latter two-step techniques, the extraction of soil P might not be exhaustive and the extraction process was terminated upon separation and removal of the extracting solution for analysis of the extracted P fraction. Factors that affected the method's efciency included the extractant's strength (i.e., weak organic acids vs. strong acids or bases), the physical force or energy expanded during the extraction process (i.e., mild vs. vigorous agitation), and the length of extraction time used for completing the extraction of soil P before the determination of P in the extract or the addition of enzymes to the extract and subsequent analysis of extracted organic P. A signicant effect was observed with the enzyme addition (i.e., EDTA-PHP fraction), especially in surface soil layers. In the 015 cm depth of the LVA soil, the EDTA-PHP fraction was, on average, 72% higher than the EEPi fraction extracted by EDTA alone. However, EDTA-PHP concentrations were not particularly high, reaching values between 10 and 16 mg P dm 3 (Fig. 1 and Tables 2 and 3). In the 0 25-cm depth, the LV soil showed a signicant increase in EDTA-PHP when compared to EEPi, with a mean increase of 183 and 220%, in CT and NT soils, respectively (Fig. 2). The highest EDTA-PHP values as a function of soil depth appeared to have shifted downward with inversion tillage and soil mixing. In the CR soil, the EDTA-PHP fraction was 67 and 85% larger than the EEPi fraction, in the control (untreated) and the manure-added CR soil, respectively. Therefore, a signicantly higher proportion of the soil total P was in organic forms in the soil developed under a temperate climatic zone. Compared to the LV soil that had similar total P content, the CR soil had by far higher concentrations of organic EDTA-PHP forms. The accessibility of these organic forms of soil P to the added phosphohydrolases also suggested an analogous mechanism by which soil microorganisms or plants obtain P to meet their nutritional needs in these soils. The hydrolytic activity occurs in the rhizosphere of plants where extracellular phosphohydrolases are released by plant roots (Duff et al., 1994; Ma et al., 2009) and microorganisms (Greiner et al., 2000; Dao and Hoang, 2008). In a review of the literature on the role of ligands in the turnover of soil organic P, extracellular phosphohydrolases were essential in letting these organisms gain access to outlying P-containing organic substrates (Dao, in press). The process is, in fact the model we use to develop the bioassay, having added fungal extracellular enzymes to soil suspensions to effect the hydrolysis of P and quantication of the soil EDTA-PHP pool (Dao, 2004; Dao et al., 2005). There are signicant implications for the management of the Oxisols, which includes optimizing the biological transformations of

applied fertilizer or the native P into organic phosphohydrolase-labile forms. No-tillage had apparently maintained the surface 015-cm depth biologically active as the LVA averaged 6% more EDTA-PHP under NT than CT management, and averaged 16% more in the surface 5 cm zone (Fig. 1). The experimental results suggested that long-term no-tillage and judicious management of phosphate fertilizers inputs could raise P saturation of the near-surface zone to benecially increase P availability; but no-tillage denitely increased biological activity in the near-surface zone to increase the enzyme-labile EDTAPHP fraction and the production of organic ligands and therefore the increased potential bioavailability of soil P in NT-LVA and LV soils (Ae et al., 1990; Dao, 2004; Rao and Dao, 2008; Dao, in press). The patterns of P availability in these three soils can also be explained more conventionally by soil composition and geochemical properties. The soil total P showed that the differences between the three soils were not as large as in labile P fractions (Fig. 4). However, the availability of the P is very low, similar to what was observed by Eberhardt et al. (2008). For example, in the 05 cm soil depth of the LVA soil, the EDTA-PHP fraction represented 2.8 and 3.4% of the total P, for the CT and NT treatments, respectively (Figs. 1 and 4). In the LV soil, the EDTA-PHP fraction represented 5.5 and 4.9% of the total P, at the same depth and tillage method. However, conventional chemical indices, such as the Mehlich 3-P soil test values, cannot detect the effect of tillage management; no signicant differences in these measurements were observed between CT and NT in both LVA and LV soils in our study (Table 4). The Mehlich 3 soil test yielded much less extracted P than the bioactive P assay used in our study, amounting to an average of 1.5 and 2.5% of the total P of LVA and LV soils, respectively (Table 4 and Figs. 1 and 2). Soil PSIM3 values were declining from the surface through the soil prole, within each land management system, directly related to the quantity of available P, as the amount of Mehlich 3-Al and Fe in the prole did not change for all three soils (data not shown). The PSIM3 values in the LVA and LV soils

Fig. 4. Effects of tillage method and phosphorus management on soil total phosphorus distribution in the 040 cm depth of LVA, LV, and CR soils. The LSD0.05 for detecting signicant differences between tillage depth treatment means were 45, 76, and 108 for the LVA, LV, and CR soils, respectively. CT = conventional tillage; NT = no-tillage; CTRL = 0 kg P ha 1; manured = 30 kg manure P ha 1.

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isotherm parameters. Meanwhile, the characterization of soil bioactive P fractions showed that there was a signicant increase in the WEP and EDTA-PHP fractions in the 015 cm depth the LVA and LV soils due to the reduction in tillage intensity. Likewise, the practice of adding organic P with land application of animal manure increased EEPi and EDTA-PHP fractions, whereas Smax did not vary between P management treatments. No-tillage has apparently maintained the near-surface zone more biologically active than CT, and has increased the accumulation of bioactive P fractions and their potential availability in NT-LVA and LV soils. Although there was a correlation between chemical indices such as the Mehlich 3-P and the EEPi estimates, the ligand-based bioactive P fractionation approach yielded an additional soil P pool, the EDTA-PHP fraction. That enzyme-labile fraction has a direct relationship to plants or microbial nutrition (Rao and Dao, 2008). Thus, the estimation of bioactive P fraction using the phosphohydrolase enzyme assay appeared to be a promising way to determine P availability in soil, and a robust methodology for diverse soils that include the Brazilian Cerrado Oxisols. Phosphorus status in such soils could prove difcult to characterize using conventional soil test methods because of the low extractability of P, and very low concentrations of labile organic forms, compared to the P-rich CR soil developed under a temperate climatic zone. Acknowledgement The authors sincerely acknowledged the technical assistance of G. Stone during the conduct of this study. References
Ae, N., Ariahara, J., Okada, K., Yoshihara, T., Johansen, C., 1990. Phosphorus uptake by pigeon pea and its role cropping systems of the Indian subcontinent. Science 248, 477480. Allmaras, R.R., Schomberg, H.H., Douglas Jr., C.L., Dao, T.H., 1999. Conservation tillage's unforeseen advantage. Resource 6, 78. Anderson, G., Williams, E.G., Moir, J.O., 1974. A comparison of sorption of inorganic phosphate and inositol hexaphosphate by six acid soils. J. Soil Sci. 25, 5162. American Public Health Association (APHA), 1998. Phosphorus: automated ascorbic acid reduction method. In: Clescerl, L.S., Greenberg, A.E., Eaton, A.D. (eds.). Standard Methods for the Examination of Water and Wastewater. Section 4500-P F, 20th ed. American Public Health Assoc., American Water Works Assoc., Water Environment Fed., Washington, DC. Anghinoni, I., Baligar, V.C., Wright, R.J., 1996. Phosphorus sorption isotherm characteristics and availability parameters of Appalachian acid soils. Comm. Soil Sci. Plant Anal. 27, 20332048. Celi, L., Presta, M., Ajmore-Marsan, F., Barberis, E., 2001. Effects of pH and electrolytes on inositol hexaphosphate interaction with goethite. Soil Sci. Soc. Amer. J. 65, 753760. Condron, L.M., Goh, K.M., Newman, R.H., 1985. Nature and distribution of soil phosphorus as revealed by a sequential extraction method followed by 31P nuclear magnetic resonance analysis. J. Soil Sci. 36, 199207. Dao, T.H., 1998. Tillage and crop residue effects on carbon dioxide evolution and carbon storage in a Paleustolls. Soil Sci. Soc. Am. J. 62, 250256. Dao, T.H., 2003. Polyvalent cation effects on myo-inositol hexakis dihydrogenphosphate enzymatic dephosphorylation in dairy wastewater. J. Environ. Qual. 32, 694701. Dao, T.H., 2004. Ligands and phytase hydrolysis of organic phosphorus in soils amended with dairy manure. Agron. J. 96, 11881195. Dao, T.H., 2007. Ligand effects on inositol phosphate solubility and bioavailability in animal manures. In: Turner, B.L., Richardson, A.E., Mullaney, E.J. (Eds.), Inositol Phosphates: Linking Agriculture and Environment. CAB International, Oxfordshire, UK, pp. 169185. Dao, T.H., in press. Extracellular enzymes in sensing environmental nutrients and ecosystem changes: Ligand mediation in organic phosphorus cycling. In: Shukla, G.C., Varma, A. (Eds.), Soil Enzymes, Chapter 5. Soil Biology series, Springer, New York, NY. Dao, T.H., Zhang, H., 2007. Rapid composition and source screening of heterogeneous poultry litter by energy dispersive X-ray uorescence spectrometry. Ann. Environ. Sci. 1, 6979. Dao, T.H., Hoang, K.Q., 2008. Dephosphorylation and quantication of organic phosphorus in poultry litter by puried phytic-acid high afnity Aspergillus phosphohydrolases. Chemosphere 72, 17821787. Dao, T.H., Codling, E.E., Schwartz, R.C., 2005. Time-dependent phosphorus extractability in calcium- and iron-treated high-phosphorus soils. Soil Sci. 170, 810821. Dao, T.H., Lugo-Ospina, A., Reeves, J.B., Zhang, H., 2006. Wastewater chemistry and fractionation of bioactive phosphorus in dairy manure. Comm. Soil Sci. Plant Anal. 37, 907924. Duff, S.M.G., Sarath, G., Plaxton, W.C., 1994. The role of acid phosphatases in plant phosphorus metabolism. Physiol. Plant. 90, 791800.

Fig. 5. Changes in adsorbed concentration of myo-inositol hexakisphosphate (mIPH) after ve desorption cycles in soil samples from the 05 cm depth of the LVA, LV, and CR soils. CT = conventional tillage; NT = no-tillage; CTRL = 0 kg P ha 1; manured = 30 kg manure P ha 1.

were considered low when compared to the results presented by Zhang et al. (2005) for 28 Oklahoma soils; Oklahoma soils had values ranging from 1.6 to 26.0%, with a mean of 3.9%. However, the calculated PSIM3 values again did not show any signicant difference between tillage method for the LVA and LV soils (Table 5).

4. Summary and conclusions As land management practices have a direct effect on the behavior and availability of soil nutrients, the effects of tillage practices or nutrient fertilization strategies on P availability was evaluated in an incubation and fractionation study of three soils with distinct biogeochemical background. Phosphate and mIPH were strongly sorbed by all three soils. Soil properties, like clay, organic matter, Al and Fe contents were related to the soil P adsorption maxima. In the 040 cm depth of the CR, LV, and LVA soils, Smax for phosphate averaged 5.5 (2.3), 5.7 ( 1.7), and 13.8 (2.7) mmol kg 1, respectively. For mIPH, Smax averaged 2.2 (0.8), 4.6 (0.7) and 5.0 (0.3) mmol kg 1 for the same depth of the CR, LV, and LVA soils, respectively. These adsorption characteristics could explain the overall differences in water-extractability of P between the three soils. However, tillage and phosphorus management effects on P availability were difcult to differentiate based solely on sorption

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