DIVISION S-4SOIL FERTILITY & PLANT NUTRITION

The Role of Particulate Organic Matter in Phosphorus Cycling

A. M. Salas, E. T. Elliott, D. G. Westfall, C. V. Cole, and J. Six* ABSTRACT
In tropical cropping systems, a better understanding of P transformations and short-term P cycling during decomposition of incorporated residues is pertinent. The primary objectives of this study were (i) to assess P immobilization in decomposing residues of sorghum (Sorghum bicolor (L.) Moench.) and crotalaria (Crotalaria juncea L.), (ii) to establish the role of soil fungi in the process, and (iii) to determine the contribution of P immobilization in explaining differences in soil P availability from plant residues in two weathered soils (Ultisol and Alfisol). Phosphorus associated with particulate organic matter (POM-P) was measured at different residue decomposition stages. Irradiated and nonirradiated residues were utilized to evaluate the origin of fungal colonization of particulate organic matter (POM). Results indicate a significant release of P from the residues at early stages of decomposition. An average of 93 and 76% of the initial POM-P was released after 5 d of decomposition in the Ultisol and Alfisol, respectively; and no significant differences were found between the residue types. After this initial 5-d period, significantly higher values of POM-P were observed for sorghum (5.8 and 7.9 mg P kg1) as compared with crotalaria (1.9 and 2.8 mg P kg1) in the two soils, suggesting a higher P immobilization in decomposing sorghum residue. Phosphorus immobilization represented up to 30% of the P in the residues and it could account for observed differences in P availability in the amended soils. A correspondence between patterns of P accumulation and fungal colonization of POM suggested that soil fungi might be responsible for this P immobilization. Further research is required however, to identify the mechanisms regulating this fungal colonization and P immobilization of plant residues in tropical acid soils.

ncorporation of legume and native plant residues in crop rotations is used to enhance nutrient availability in many tropical agroecosystems. The ability of plant residues to supply nutrients to subsequent crops is of special interest in the management of soil fertility in tropical areas, allowing the possibility of reducing fertilizer dependency. Much attention has been devoted to determine plant, biological, and environmental factors influencing the release and mineralization of N from plant residues (Vanlauwe et al., 1996; Bending et al., 1998). However, relatively few studies have evaluated the in-

a, Facultad de Agronom a. UniversiA.M. Salas, Instituto de Edafolog dad Central de Venezuela. Maracay, Apdo. Postal 4579. Venezuela; E.T. Elliott, School of Natural Resources, University of Nebraska, Lincoln, NE 68583; D.G. Westfall, Soil and Crop Dep., Colorado State University, Fort Collins, CO 80523; C.V. Cole, Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523; J. Six, Dep. of Agronomy and Range Science, University of California, Davis, CA 95616 and Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523. Received 20 July 2001. *Corresponding author (jwsix@ucdavis.edu). Published in Soil Sci. Soc. Am. J. 67:181189 (2003).

corporation of plant residues on P cycling, and its contribution to plant nutrition (Dalal, 1979; McLaughlin et al., 1988a; Umrit and Friesen, 1994). Of these studies, most have focused on chemical reactions, particularly the effect of plant residues on P sorption and inorganic P fractions of the soil (Iyamuremye et al., 1996; Nziguheba et al., 1998). Few have considered biological transformations associated with the release of P from residues, and subsequent accumulation and turnover of organic P during decomposition (McLaughlin et al., 1988c). Increasing amount of residue and litter in the topsoil results in accumulation of organic P, which makes soil P availability less predictable by chemical extraction methods for inorganic P (Bowman and Halvorson, 1997). Utilization of conventional P fractionation procedures based on solubility of different P compounds may not discriminate among organic P pools differing in turnover. Conventionally available inorganic P soil testing may not properly assess the potential contribution of residue P and P transformations following the decomposition of residues. Availability of P from plant residues is affected by the initial release of P from the residues and microbial transformations occurring during decomposition of plant residues (Blair and Boland, 1978). Use of radioactive tracers (32P and 33P) revealed that P release from residues is relatively rapid (Friesen and Blair, 1988). Phosphorus immobilization by microorganisms, turnover of microbial P, and mineralization of microbial byproducts seem to be the major processes regulating P cycling and P availability from plant residues (McLaughlin et al., 1988b). Recent decomposition studies using litter bags indicated that a significant part of microbial immobilization of P takes place inside decomposing plant residues (Schomberg and Steiner 1999; Mafongoya et al., 2000) where microbial turnover and P mineralization might differ from the processes taking place in the bulk soil. Phosphorus immobilization in decomposing residues is believed to be responsible for as much as a 70% increase in the amount of P in agricultural residues (Tian et al., 1992; Schomberg and Steiner, 1999), and from 20 to 200% increase in tree leaves (McTiernan et al., 1997; Conn and Dighton, 2000). Some reports suggested that immobilization of P in residues could significantly decrease P availability from added plant residues (Schomberg and Steiner, 1999; Mafongoya et al., 2000). The role of soil fungi in the immobilization of nutrients in decaying litters has been mentioned in previous
Abbreviations: OM, organic matter; POM, particulate organic matter; POM-C, C associated with the POM; POM-N, N associated with POM; POM-P, P associated with POM; PT, polytungstate.

181

182

SOIL SCI. SOC. AM. J., VOL. 67, JANUARYFEBRUARY 2003

studies. Nitrogen immobilization in many different types of litter has been attributed to the translocation of the element from the soil inorganic pool by soil fungi (Hart et al., 1993; Bedrock et al., 1998; Frey et al., 2000). A similar mechanism has been suggested to explain the immobilization of P in decaying litters. In tree litters, it has been found that immobilization of P can closely follow hyphal colonization of the litter (Conn and Dighton, 2000). Similarly, immobilization of P in plant residues has been shown to occur in agroecosystems (Friesen and Blair, 1988; Tian et al., 1992; Schomberg and Steiner 1999). However, no evidence has been presented regarding the role of soil fungi and the factors influencing this process in agricultural systems. This lack of information can be partially because of difficulties in sampling decomposing residues in agricultural systems. The fact that in most agricultural systems, plant residues are in contact with the soil, limits the utilization of litter bags to estimate the P accumulation in residues partially or completely incorporated into the soil. The presence of soil contaminants, even for residues applied to the surface, has been shown to produce high variability in the amount of P accumulated in litter bags (Schomberg and Steiner, 1999). Particulate organic matter has been considered an active organic matter (OM) pool that participates in the release of nutrients in cultivated areas (Cambardella and Elliott, 1992; Magid et al., 1996). Following POM dynamics rather than using the litter bag approach is more realistic, allowing evaluations of residues that are in contact with soil particles. Recent studies have indicated that dynamics of this macroorganic matter can significantly influence P cycling in crop rotations with either legumes or natural fallow (Maroko et al., 1999; Vanlauwe et al., 2000), suggesting that P contained in this OM pool can have a significant influence on P availability. Changes in P associated with the POM could provide valuable information about the pattern of release of P from plant residues and P immobilization in decomposing litters. The main objectives of this study were: (i) to evaluate changes in P associated with POM during decomposition of plant residues, (ii) to establish
Table 1. Selected characteristics of soils utilized in the incubation studies.
Soil characteristics Taxonomic group Texture clay, g kg1 soil sand, g kg1 soil silt, g kg1 soil pH (1:1, soil/water) Total organic C, g kg1 Resin extractable Pi, mg P kg1 Bray I extractable P, mg P kg1 Total P, mg P kg1 Sorbed P from isotherms, mg P kg1 Total cation-exchangeable capacity, NH4AcO pH 7.0, cmolckg1 Exchangeable Al in KCl, cmolckg1 Ultisol Typic Haplustult 50 900 50 5.8 6.5 2.2 7.5 155 1.7 3.1 0.05 Alfisol Typic Paleustalf 120 700 180 4.8 8.7 0.5 7.8 285 40 5.8 0.7

the role of soil fungi in the immobilization of P in decomposing residues, and (iii) to determine the contribution of this P immobilization to differences in short-term P availability from these plant residues. Plant materials having the same C/P ratio, but from different species, were utilized to evaluate the influence of some plant factors in determining P immobilization dynamics in decomposing residues. MATERIALS AND METHODS Soil and Plant Materials
Incubation experiments were conducted using the top layer (015 cm) of two weathered soils (Alfisol and Ultisol) representative of semiarid agricultural areas located in the central and eastern part of Venezuela. Selected soil characteristics are summarized in Table 1. Greenhouse grown residues of sorghum and crotalaria were utilized to conduct decomposition studies under controlled conditions. Leaves and stalks were harvested 60 d after planting. Plant materials were airdried and cut in 1-cm pieces before being ground (1000 m) using a Wiley grinder (Thamas-Wiley, Swedesboro, NJ). The percentage of material retained between the 500- and 1000m sieve was 85 and 90% for the sorghum and crotalaria residues, respectively. Analyses were performed to characterize the plant materials. Total C and N in finely ground samples were determined by dry combustion using a LECO CHN-1000 analyzer (Leco Corp., St. Joseph, MI). Total P was determined colorimetrically (Murphy and Riley, 1962) after HNO3HClO4 digestion. Extractable inorganic residue P was measured after cold water extraction for 1h (150 mg of air-dried plant sample and 25 mL of distilled water) and filtration with Whatman 42 filter paper (Whatman, Maidstone, UK). The same cold water extract was used to determine the total amount of dissolved organic C, using an infrared Shimadzu TOC-500 analyzer (Shimadzu, Lenexa, KS) after acidification. Residue cellulose and lignin contents were determined on ground samples using the acid detergent fiber method (Van Soest, 1967). Selected residue characteristics are shown in Table 2.

Separation of Particulate Organic Matter for Phosphorus Analysis

A series of preliminary experiments was conducted to evaluate different methods to separate the POM for P analysis. Conventional methods for obtaining POM are based on the utilization of sodium hexametaphosphate (NaPO3) for dispersion of the soil aggregates (Cambardella and Elliot, 1992), which make them unsuitable for P analysis. Alternative methods include the utilization of distilled water (Maroko et al., 1999), Na-exchange resins (Dalal and Mayer, 1986), and Na2CO3 solution (Vanlauwe et al., 1998). Two aspects, however, limit the utilization of these methods for studies of
Table 2. Selected characteristics of the plant residues utilized in the incubation studies.
Residue type Plant characteristic C/N ratio Total P, g P kg1 plant Water extractable Pi, % of total plant P C:P ratio cellulose, g kg1 plant lignin, g kg1 plant Water extractable organic C, g C kg1 plant Sorghum 16 1.31 73 332 330 46 117 Crotalaria 18 1.23 63 336 470 79 57

Resin extractable inorganic P determined using resin strips according to Parfitt et al. (1994). Sorbed P required to maintain a soil solution P concentration of 0.2 g P mL1, obtained from isotherms according to the Fox and Kamprath (1970) method.

SALAS ET AL.: PARTICULATE ORGANIC MATTER AND PHOSPHORUS CYCLING

183

POM-P dynamics. The first is the incomplete dispersion of aggregates, which affects the recovery of POM by sieving or flotation procedures; and second, the potential solubilization of organic P compounds contained in the POM when using alkaline solutions (Na2CO3). Results of preliminary tests indicated that a 0.05 M NaCl solution effectively dispersed the soil aggregates with C recoveries (72 to 92%) similar to those obtained by dispersion with sodium hexametaphosphate (Salas, 2001); therefore, the NaCl solution was chosen for separation of the POM. The procedure for POM separation was: 25 g of moist soil sample was weighed in triplicate into 50-mL plastic containers. Thirty milliliters of a 0.05 M NaCl solution was added. The soil suspension was shaken for 2 h in a rotatory shaker at 150 rpm, after which it was passed through a 53-m sieve and rinsed with distilled water until a clear solution was obtained. Material retained in the sieve, a mixture of sand and POM, was kept for further separation by flotation. The soil suspension containing soil particles and OM 53 m was discarded. A modified method for density flotation of POM was adapted from Six et al. (1998). Briefly, flotation of the POM was done by transferring the material retainedon the sieve to a 50-mL graduated conical centrifuge tube containing 35 mL of Na polytungstate (PT) (density 1.85 Mg m3). The suspended material was mixed by slow reciprocal shaking by hand (10 strokes) and then slowly placing the centrifuge tube in the vertical position. Following centrifugation at 1250 g for 45 min, the material was aspirated onto a 20-m mesh nylon filter, rinsed throughly with distilled water to remove the excess of NaPT, and then transferred to a preweighed aluminum pan to be dried at 60C for 24 h. Sodium polytungstate was recycled using the method suggested by Six et al. (1999). The dried POM was weighed and ground by hand using a porcelain mortar and pestle.

P analysis using the colorimetric blue method (Murphy and Riley, 1962). The amount of C, N, and P contained in the POM (POM-C, POM-N, and POM-P) per unit of soil dry weight was determined for both plant residue treatments taking into account the soil water content and the POM content of the soil. At the end of the experiment soil samples were also analyzed for P availability. Strongly sorbed P was extracted using air-dried soil samples and the Bray (0.03M NH4F 0.025M HCl) solution as described in Olsen and Sommers (1982). Organic and inorganic P fractions were obtained by sequential P fractionation (Hedley et al., 1982). Total concentration of P in the extracts was determined by inductively coupled plasma, while inorganic P was analyzed colorimetrically by the molybdate blue method (Murphy and Riley, 1962).

Fungal Colonization of Particulate Organic Matter

A second study was conducted to investigate the role of soil fungi in the immobilization of P in decomposing plant residues. Fungal colonization of the POM was determined at different stages of decomposition of crotalaria and sorghum residues. Irradiated and nonirradiated plant residues were used to establish the contribution of the plant inoculum to the fungal colonization of the plant residues. Irradiation was performed by exposing plant materials to radiation (2.5 104 Gy) for 16 h. Five treatments resulted from the utilization of two residue types, irradiated versus nonirradiated treatments, and a control (without residue). Preparation of experimental units was similar to the previous experiment, except that in this case only the Alfisol was used. Soil sampling to obtain the POM occurred after 0, 5, 15, 30, and 45 d of incubation. Separation of the POM from the soil was performed as previously indicated, and POM samples were used for further analysis of fungal colonization. To estimate the fungal colonization of decomposing residues, the fungi growing in fresh POM samples was stained using the trypan blue (C34H24N6Na4O14S4) method (Phillips and Hayman, 1970), initially developed to assess parasitic and vesicular-arbuscular mycorrhizal infection in roots. In contrast to the original method, we used a lower concentration of KOH (0.45 M instead of 1.80 M ) and a shorter period for the clearing pretreatment (10 instead of 30 min). The POM pieces cleared with the base during 10 min at 90C were carefully washed with distilled water and immersed in 0.1 M HCl for 1 h. After this period, the POM pieces were stained with trypan blue (0.05%) in lactophenol at 60C for 15 min. The stained POM samples were then placed in destaining solution (lactic acid/ distilled water/glycerine, 1:1:2; v/v) to remove excess trypan blue and were transferred to microscope slides for observation under the microscope at 50 magnification. Images of the microscopic field were captured using the Optronics cooled CCD camera (Model DEI-470, Diagnostics Instruments, Inc., Sterling Heights, MI) and Adobe Photoshop image capturing software (Adobe Systems Inc., San Jose, CA). Visible hyphal width and length, as well as POM area, were measured in each POM piece using the IPLab Spectrum image analysis software (Signal Analytics Corp., Vienna, VA). Since no significant correlation (r 2 0.08) was found between POM area and fungal length, fungal colonization was calculated by dividing the hyphal length by the POM area. A sample size of 20 pieces of fresh POM was established based on preliminary calibration tests conducted to quantify the variability in fungal colonization. After sampling for fungal staining, the remaining POM was dried at 60C and its weight recorded. Fungal colonization on a POM weight basis was estimated by taking into account the average weight of six sets of 20 (dried) POM

Phosphorus Dynamics in Particulate Organic Matter

To evaluate P dynamics in the POM a decomposition study was conducted using crotalaria and sorghum residues in the two soils. For each soil, experimental units were prepared by thoroughly mixing of 40 g of air-dried soil (sieved to 4 mm) with each of the two plant materials (20 g kg1 soil). The incorporation rate of 20 g kg1 soil was based on biomass production (13 Mg ha1 yr1) under field conditions and an assumed incorporation of this biomass into the upper 5-cm soil layer with a bulk density of 1.3 g cm3. Controls without plant residues were also prepared. Treatments were replicated three times and six sampling times were used: 0, 5, 15, 30, 45, and 60 d after incubation. Soil with or without added plant residue were incubated in specimen cups placed in Mason jars at a constant temperature of 25C. A modified Hoaglands nutrient solution (Hoagland and Arnon, 1938) containing macro and micronutrients (except P) was uniformly applied, using a syringe, to bring the soil moisture to 65% of the water holding capacity; or 15 and 20% (w/w), for the Ultisol and Alfisol, respectively. Amount of residue total P added was 24.6 and 26.0 mg P kg1 soil, for the crotalaria and sorghum residues. Treatments and their replicates were randomly assigned within each sampling time and experimental units were destructively sampled for separation and analysis of POM at each sampling date. Moist soil samples were dispersed, and the POM separated from the sand by flotation using NaPT, as described previously. Total C and N in dried POM subsamples were determined by dry combustion using a LECO CHN1000 analyzer (Leco Corp., St. Joseph, MI). A POM subsample of 250 mg was also used for HNO3HClO4 digestion and total

184

SOIL SCI. SOC. AM. J., VOL. 67, JANUARYFEBRUARY 2003

pieces. Previous results indicated a low variability in the weight of 20 POM pieces, with coefficient of variations 20% for all sampling times. Estimates of fungal colonization on a soil weight basis were also obtained, taking into account the POM content and the water content of the soil samples. Finally, fungal biomass C in the POM was estimated by multiplying fungal biovolume by the specific C content of the fungi (0.132 Mg C m3) as suggested by Bloem et al. (1995). The experimental data were analyzed as a completely randomized two-way factorial design. Within soil, treatments and sampling times were considered factors. Data for fungal colonization as well as fungal biomass in the POM were logtransformed prior to analysis to meet normality and homogeneity of variance requirements for ANOVA. Mean comparisons were performed at each sampling time using the Tukeys Honestly Significant Difference (P 0.05) calculated by the GLM procedure of the SAS program (SAS Institute, 1990).

RESULTS AND DISCUSSION Phosphorus Dynamics in Particulate Organic Matter

Phosphorus associated with POM changed rapidly during residue decomposition and its dynamics significantly differed from that observed for C and N contained in the POM. The amount of P contained in the POM at the beginning of the incubation (Day 0) represented about 25% of the total P added in both residues. After 5d of decomposition a further significant decline in POM-P occurred, indicating a rapid release of P from residues in both soils (Fig. 1). In the Ultisol, 5 d of decomposition resulted in a release of 92 and 95% of the P contained in the POM at Day 0 from the sorghum and crotalaria residues, respectively. In the Alfisol, 75 and 77% of the P contained in the POM at Day 0 was released during this 5-d period. This significant loss of

P from the residues contrasted with a lower proportion of C and N released from the POM during the same period. A maximum of 18% of C and 59% of N were released from the POM after 5 d of decomposition (Table 3). These results suggest some differences in the mechanism of release of C and N from plant residues. While initial release of C and N from the residues seemed to be more controlled by the utilization of the substrates by the microbes, the release of P was very rapid and relatively independent of the utilization of C by the microbes. These results can be partially explained by a large proportion of the plant P present in inorganic forms and the ability of the organic P compounds to be mineralized by plant enzymes. Previous studies have shown that because of autolysis of the plant cells and hydrolysis of phosphate esters by plant phosphatases, a significant amount of P can be released from dried plant materials even without microbial activity (Martin and Cunningham, 1972). In this study, 73 and 63% of the total plant P was in inorganic P forms in the sorghum and crotalaria residues, respectively (Table 2). Previous studies have also indicated a rapid release of residue P at early stages of decomposition. For example, Friesen and Blair (1988) observed that more than 50% of residue-derived P was in inorganic soil pools after 11 d of incorporation. McLaughlin et al. (1988c) also found that 50% of the 33P applied in labeled residues of Medicago lupulina was extractable from the soil with NaHCO3 (pH 8.5) at the beginning of the experiment. They estimated that the amount of P remaining in the residues after 15 d of decomposition accounted for only 4% of the 33P initially added in the residues. They attributed the rapid release of P from the residues to the high proportion of inorganic P initially present in the residues

Fig. 1. Changes in P associated with the particulate organic matter (POM-P) after the incorporation of plant residues in the Ultisol and Alfisol. (Bars represent Tukeys HSD (P 0.05) for mean comparisons between plant residue treatments within sampling time. Data points are means of three replicates with bars representing their standard errors.)

SALAS ET AL.: PARTICULATE ORGANIC MATTER AND PHOSPHORUS CYCLING

185

and rapid microbial utilization of readily available inorganic P at early stages of decomposition. After the initial rapid release of P from the residues, an increase in POM-P was observed from 5 to 60 d of incubation in both soils (Fig. 1). This increase in POM-P was strongly dependent on the plant residue (P 0.001) in both soils. In the Ultisol, the POM-P increased from 0.5 to 5.8 mg P kg1 soil for the sorghum residue; while for crotalaria, the value changed from 0.4 to 2.8 mg P kg 1 soil. For the same period of time in the Alfisol, the POM-P changed from 1.5 to 7.9 mg P kg1 soil and from 1.8 to 1.9 mg P kg1 soil for sorghum and crotalaria residues, respectively. Thus, the amount of POM-P in the sorghum treatment increased 13 and 5 times in the Ultisol and Alfisol, respectively; POM-P in the crotalaria treatment increased eight times in the Ultisol and a very small increase was observed in the Alfisol. Particularly important to note is that in both soils, the amount of sorghum-derived POM-P was at the end of the experiment nearly the same as that at the beginning of the experiment (Fig. 1). These results indicate that a significant amount of plant P can be reincorporated in decomposing residues at later stages of decomposition, but amount varies with residue type. Increasing sorghum-derived POM-P also corresponded with an increase in the amount of N accumulated in the POM (POM-N) after 5 d of incubation (Table 3). A significantly higher amount of POM-N was found at the end of the experiment with sorghum as compared with crotalaria, even though both residues had similar amounts of POM-N at the beginning of the experiment (Day 0). Although it was not possible to differentiate the N that was originally in the POM from that immobilized in the POM during decomposition, the data show a significant amount of N remaining in the sorghum-derived POM at
Table 3. Changes in C and N associated with the particulate organic matter (POM-C, POM-N) during decomposition of plant residues in two weathered soils from the tropics.
Residue type Days of incubation 0 5 15 C kg1 soil 5485 a 5710 a 955 N kg1 soil 271 a 124 b 63 C soil 5401 a 5349 a 712 N kg1 soil 272 a 161 b 55 kg1 30 45

the end of the experiment (109 and 81%, for the Ultisol and Alfisol, respectively), suggesting that immobilization of N occurred in this residue (Table 3). In the crotalaria, by contrast, there was a significantly lower amount of POM-N remaining at the end of the incubation (60 and 37% of the initial POM-N, for the Ultisol and Alfisol, respectively). This corresponds with the previous observation showing a larger increase in sorghum-derived POM-P compared with crotalaria-derived POM-P. Increasing POM-P led us to hypothesize that an immobilization of P in the litter might have occurred as microorganisms start utilizing more recalcitrant C sources in decomposing litters and the microbial demands for P were increased. If this hypothesis were true, it would be expected that differences in the amount of P accumulated in the POM could be explained by differences in the demands for P of the microbial communities colonizing the decomposing litters. To test this hypothesis, an experiment was conducted to determine fungal colonization of the POM during decomposition of both residues in the Alfisol.

Fungal Colonization of Particulate Organic Matter

Fungal colonization of the decomposing litter was strongly influenced by residue type (P 0.001). From early stages of decomposition, sorghum residues had consistently higher levels of fungal colonization measured by fungal length per unit of POM weight (Table 4). Because of the greater fungal length and the larger width of the hyphae colonizing the sorghum residue (Salas, 2001), fungal biomass in this litter significantly exceeded that obtained in the crotalaria residues (Table 4). Peak fungal biomass occurred at 30 d of incubation in which 35.3 and 0.2 mg fungal C kg1 soil were found for sorghum and crotalaria POM, respectively. The sequence of fungal colonization presented in Table 4 agrees with previous research indicating that fungi can colonize plant residues at later stages of decomposition, given their competitive ability to utilize more resistant C compounds (Beare et al., 1992).
Table 4. Fungal colonization and fungal biomass of particulate organic matter (POM) during decomposition of irradiated and non irradiated plant residues in the Alfisol.
Days of incubation Treatment 5 15 g 1 30 45 80.1 77.1 7.8 6.7 26.3 25.9 0.1 0.1 a NS b NS a NS b NS

Sorghum crotalaria control Sorghum crotalaria control

Sorghum crotalaria control Sorghum crotalaria control

ULTISOL POM-C, soil basis, mg 7248 a 6670 a (8) 7768 a 6390 a (18) 1013 1021 POM-N, soil basis, mg 273 a 233 a (15) 254 a 104 b (59) 71 73 ALFISOL POM-C, soil basis, mg 8077 b 7471 a (8) 9220 a 7695 a (17) 1263 707 POM-N, soil basis, mg 332 a 190 a (43) 348 a 223 a (36) 66 50

3790 b 4722 a 899 278 a 154 b 59

3055 b 4025 a 910 297 a 153 b 61

5224 a 5375 a 886 288 a 137 b 58

3815 b 4666 a 737 269 a 128 b 41

POM Fungal colonization, m Nonirradiated sorghum 60.8 a 83.0 a 151.0 a Irradiated sorghum 45.6 ** 66.6 * 152.7 NS Nonirradiated crotalaria 1.9 b 2.7 b 11.7 b Irradiated crotalaria 2.3 NS 2.4 NS 8.4 NS Fungal biomass associated with POM, mg C kg1 soil Nonirradiated sorghum 18.4 a 19.4 a 35.3 a Irradiated sorghum 12.1 * 16.2 NS 38 NS Nonirradiated crotalaria 0.1 b 0.1 b 0.2 b Irradiated crotalaria 0.1 NS 0.1 NS 0.1 NS

Treatment means followed by the same letter within soil and sampling time are not statistically different according to the Tukeys mean test (P 0.05). Values in parentheses represent percentage of the initial POM-C and POM-N lost in the first five days of decomposition.

* Significance at the 0.05 probability level. ** Significant at the 0.01 probability level. Means of nonirradiated plant residues followed by the same lower case within each sampling time are not significantly different according Tukeys test (P 0.05). NS indicates no significant difference (P 0.05).

186

SOIL SCI. SOC. AM. J., VOL. 67, JANUARYFEBRUARY 2003

Fig. 2. Fungal colonization of the particulate organic matter (POM) observed under the microscope after (a) 15 and (b) 30 d of decomposition of sorghum residue in the Alfisol.

Visual differences in fungal colonization of the POM at two stages of decomposition of the sorghum residues are shown in Fig. 2. These results of fungal colonization in the POM are in the range previously described for decomposing litters (30200 m g1) using the litter bag approach (Conn and Dighton, 2000). It is important to note, however, that fungal colonization could have been underestimated, since fine hyphae with widths of 2.0 m cannot be seen under a magnification of 50. Close examination of the POM at higher magnification (200) revealed the existence of fine hyphae (width 1.5 m), especially at the edges of the POM. Although the contribution of these hyphae was not assessed in this study, it is believed, based on the influence of width in the biovolume calculations, that their biomass was relatively small. The origin of fungi colonizing the litters was further investigated by the utilization of irradiated plant residues. In the sorghum residue, the irradiated residue had significantly lower fungal colonization than the nonirradiated residue at the beginning of the experiment (5 and 15 d of incubation). However, after that initial period, no

differences were found between the irradiation treatments (Table 4). This indicates that a fraction of the initial fungal colonization in the sorghum residue can be attributed to the fungal inoculum present in the residue but after that initial period, soil fungi became the dominant group in the litter. In the crotalaria residue, the irradiation treatment did not affect fungal colonization of the POM suggesting that a low inoculum level existed in this residue. A strong correspondence between the increase in POM-P (Fig. 1) and the pattern of fungal colonization shown in Table 4 suggests that immobilization of P in these partially decomposed plant residues might be mediated by soil fungi. Immobilization of P and many other nutrients in decomposing plant residues has been largely suggested as a mechanism to facilitate the establishment of microbial communities and the decomposition of litters in forests and agroecosystems (Melillo and Aber, 1984). Studies have shown that microbial immobilization of N and P from the mineral pool to litter can be substantial (Tian et al., 1992; Hart et al., 1993; Schomberg and Steiner, 1999, Frey et al., 2000). The possible role of soil fungi in this immobilization has been attributed to the ability of fungi to colonize more recalcitrant and nutrient-poor substrates, given their efficient utilization of C substrates (Sakamoto and Oba, 1994) and ability to translocate nutrients from spatially separated sources. Identification of N compounds of fungal origin in decomposing wheat (Triticum aestivum) straw by cross-polarization magic-angle spinning 15N nuclear magnetic resonance (Bedrock et al., 1998) as well as experiments of fungal-exclusion using 15N (Frey et al., 2000) have confirmed the participation of fungi in the mobilization of N into decomposing plant residues. The pattern of changes in POM-P, which were characterized by a rapid depletion of P followed by a consistent increase in the accumulation of P, suggests that most of the P in decomposing residues was immobilized during decomposition, and relatively less P remained from the original plant P. Assuming that microbial immobilization was the main mechanism of P transport into the residue, it would be expected that most of the P accumulated in the decomposing residues was microbially derived. However, estimates of P immobilization in the sorghum residue based on fungal biomass in the POM (Table 4) and the P content of fungal cells indicate that allocation of P in the fungal biomass can only account for a small fraction of the total P accumulated in the POM by the end of the experiment. A fungal biomass of 26.3 mg C kg1 soil (58 mg kg1 soil) for the sorghum treatment at 45 d of incubation in the Alfisol, and a P content of the fungal cells varying from 2 to 14 g P kg1 (Brookes et al., 1982; Hedley and Stewart, 1982) could theoretically account for only 0.1 to 0.8 mg P kg1 soil immobilized in the fungal biomass. These values are significantly lower than 5.3 mg P kg1 soil, the observed amount of P accumulated in the sorghum-derived POM over the same period of time (Fig. 1). This suggests that a large proportion of the P in the decomposing residues was composed of microbial residual products that had been accumulated in the residue during decomposition.

SALAS ET AL.: PARTICULATE ORGANIC MATTER AND PHOSPHORUS CYCLING

187

A slower rate of P mineralization in these microbial products relative to the turnover rate of microbial P could explain the accumulation of microbially derived P in the residues. Studies of P cycling in microcosms using 32P-labeled bacterial cells (Barsdate et al., 1974) have indicated that turnover of microbial P is greater than P mineralization from microbial detrital materials. This study supports previous reports indicating an accumulation of P in decomposing plant residues, and highlights the possible role of soil fungi in this immobilization process. As plant residues become rapidly depleted in P it is believed that the soil available P pools supply the P demands of fungi colonizing more recalcitrant substrates. Since no other source of P was added with the plant residues, desorption of P from the soil surrounding the decomposing residues must have been the main source of available P for the fungi growing in the litter. The ability of microorganisms to effectively utilize P sorbed on Fe and Al minerals has been recognized using culture media isolated from weathered soils (Shang et al., 1996; He and Zhu, 1998). Abiotic immobilization of P, that is, incorporation of inorganic P into organic compounds by chemical reactions, as well as transfer of P into the residues by diffusion, cannot be discarded as mechanisms of P transport into decomposing residues. We speculate, however, that the contribution of these mechanisms would be small, given the low accumulation of P in the residue having low fungal colonization and the possibility that any inorganic P transported inside the residue by diffusion would be removed during the POM separation procedure. Immobilization of P into POM might also indicate that a proportion of P added as plant residues could eventually be retranslocated into the litter at later stages of decomposition, resulting in a decreased P availability for plant uptake. Considering the amount of P located in the POM at the end of the experiment (Fig. 1) and the total P added as plant residues in both soils, it is estimated that between 8 and 30% of the P added in the residues can be incorporated in the litters via microbial immobilization. The amount of P immobilized in the sorghum (5.5 and 7.9 mg P kg 1 soil for Ultisol and Alfisol, respectively) and crotalaria residue (2.6 and 1.9 mg P kg1 soil for Ultisol and Alfisol, respectively) at the end of the experiment (Fig. 1), could account for

observed differences in P availability between these residues. The sorghum residue had higher immobilization of P in the litter and consistently lower levels of available P in both soils, indicated by results of Bray extractable P and inorganic P fractions Pi(resin NaHCO3) and PiNaOH (Table 5). This negative relationship between P immobilization in the litter and available soil P suggests that immobilization of P in the residues affect the turnover of microbial P and the subsequent mineralization of microbial residual products. Results indicating a comparatively larger amount of organic P extracted with NaOH (Po-NaOH fraction, in Table 5) for the sorghum residue in both soils, effectively confirmed an accumulation of organic P and consequently, a lower P mineralization in the sorghum residue. Previous results using the same plant materials have also indicated a significant decrease in the rate of microbial P losses for sorghum amended soil and comparatively a lower P mineralization (Salas, 2001). The fact that these residues had approximately the same (low) C/N and C/P ratios indicates that some other plant quality factors influenced the microbial colonization and consequently, the immobilization of P in the residues. Several plant biochemical factors have been suggested to affect soil microbial communities. Soluble C compounds, C/N ratio, the presence of inhibitors, and the contents of cellulose and lignin have been identified as affecting microbial activity during decomposition (Swift et al., 1979). A larger amount of easily decomposable substances and a lower content of lignin and cellulose in the sorghum residue, as compared with crotalaria (Table 2), may have contributed to the colonization of this residue. At early stages of decomposition, the presence of soluble C compounds may have favored the development of some species of sugar fungi (Griffiths et al., 1999). At later stages of decomposition, after soluble C compounds had been consumed, fungi may have had a better ability to colonize less recalcitrant substrates present in the sorghum residue. Utilization of recalcitrant sources in the sorghum residue could also have been enhanced by a priming effect produced by the decomposition of more readily decomposable C, as suggested by Vanlauwe et al. (1994). In contrast, the more recalcitrant substrates in the crotalaria residue

Table 5. Phosphorus availability measured by Bray extraction and sequential P fractionation procedures for the different soils and plant residue treatments at the end of the experiment.
Treatments P Bray Pi(resin NaHCO3) ULTISOL Sorghum Crotalaria Control HSD0.05 Sorghum Crotalaria Control HSD0.05 7.2 16.0 7.5 2.8 9.2 15.4 7.8 2.9 5.9 11.8 6.3 1.7 ALFISOL 6.7 9.1 3.6 1.3 5.8 4.5 4.5 2.2 19.1 25.4 14.9 2.8 27.7 21.1 19.2 2.1 1.2 1.5 2.7 1.5 7.3 14.7 8.4 2.3 15.1 6.9 8.9 1.5 PoNaHCO3 mg P kg1 soil PiNaOH PoNaOH

Pi inorganic phosphorus. Po organic phosphorus.

188

SOIL SCI. SOC. AM. J., VOL. 67, JANUARYFEBRUARY 2003

probably limited substrate availability, and affected the fungal colonization. The influence of microbial inhibitors, such as water-soluble phenolic acids and polyphenols, in shaping microbial communities during decomposition of plant residues (Palm and Sanchez, 1991) cannot be disregarded since no information was available about the content of these compounds in the sorghum and crotalaria residues.

REFERENCES
Barsdate, R.J., R.T. Prentki, and T. Fenchel. 1974. Phosphorus cycle of model ecosystem: Significance for decomposer food chains and effect of bacterial grazers. Oikos 25:239251. Beare, M.H., R.W. Parmelee, P.F. Hendrix, W. Cheng, D.C. Coleman, and D.C. Crossley, Jr. 1992. Microbial and faunal interactions and effects on litter nitrogen and decomposition in agroecosystems. Ecol. Monogr. 62:569591. Bedrock, C.N., M.V. Cheshire, B.L. Williams, I. Solntseva, S.J. Chapman, J.A. Chudek, and B.A. Goodman. 1998. Identification of nitrogenous components of fungal and bacterial origin immobilized in decomposing wheat straw by NMR spectroscopy using 15N CPMAS. Soil Biol. Biochem. 30:113115. Bending, G.D., M.K. Turner, and I.G. Burns. 1998. Fate of nitrogen from crop residues as affected by biochemical quality and the microbial biomass. Soil Biol. Biochem. 30:20552065. Blair, G.J., and O.W. Boland. 1978. The release of phosphorus from plant material added to soil. Aust. J. Soil Res. 16:101111. Bloem, J., P.R. Bohhuis, M.R. Veninga, and J. Wieringa. 1995. Microscopy methods to estimate biomass and activity of soil bacteria and fungi. p. 162172. In K. Alef and P. Nannipieri (ed.) Methods in applied soil microbiology and biochemistry. Academic Press, London. Bowman, R.A., and A.D. Halvorson. 1997. Crop rotation and tillage effects on phosphorus distribution in the Central Great Plains. Soil Sci. Soc. Am. J. 61:14181422. Brookes, P.C., D.S. Powlson, and D.S. Jenkinson. 1982. Measurement of microbial biomass phosphorus in soil. Soil Biol. Biochem. 14: 319329. Cambardella, C.A., and E.T. Elliott. 1992. Particulate soil organic matter changes across a grassland cultivation sequence. Soil Sci. Soc. Am. J. 56:777783. Conn, C., and J. Dighton. 2000. Litter quality influences on decomposition, ectomycorrhizal community structure and mycorrhizal root surface acid phosphatase activity. Soil Biol. Biochem. 32:489496. Dalal, R.C. 1979. Mineralization of carbon and phosphorus from carbon-14 and phosphorus-32 labelled plant material to soil. Soil Sci. Soc. Am. J. 43:913916. Dalal, R.C., and R.J. Mayer. 1986. Long-term trends in fertility of soils under continuous cultivation and cereal cropping in Southern Queesland. III Distribution and kinetics of soil organic carbon in particle-size fractions. Aust. J. Soil Res. 24:293300. Fox, R.L., and E.J. Kamprath. 1970. Phosphate sorption isotherms for evaluating the phosphate requirements of soil. Soil Sci. Soc. Am. Proc. 34:902907. Frey, S.D., E.T. Elliott, K. Paustian, and G.A. Peterson. 2000. Fungal translocation as a mechanism for soil nutrient inputs to surface residue decomposition in a no-tillage agroecosystem. Soil Biol. Biochem. 32:689698. Friesen, D.K., and G.M. Blair. 1988. A dual radio tracer study of transformations of organic, inorganic and plant residue phosphorus in soil in the presence and absence of plants. Aust. J. Agric. Res. 26:355366. Griffiths, B.S., K. Ritz, N. Ebblewhite, and G. Dobson. 1999. Soil microbial community structure: Effect of substrate loading rate. Soil Biol. Biochem. 31:145153. Hart, S.C., M.K. Firestone, E.A. Paul, and J.L. Smith. 1993. Flow and fate of soil nitrogen in an annual grassland and a young mixedconifer forest. Soil Biol. Biochem. 25:431442. He, Z., and J. Zhu. 1998. Microbial utilization and transformation of phosphate adsorbed by variable charge minerals. Soil Biol. Biochem. 30:917923. Hedley, M.J., J.W.B. Stewart, and B.S. Chauhan. 1982. Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Sci. Soc. Am. J. 46:970976. Hedley, M.J., and J.W.B. Stewart. 1982. Method to measure microbial phosphate in soils. Soil Biol. Biochem. 14:377385. Hoagland, D.R., and D.I. Arnon. 1938. University of California Agricultural Experimental Station Circular #347. University of California, Davis, CA. Iyamuremye, F.R., R.P. Dick, and J. Baham. 1996. Organic amendments and phosphorus dynamics: Distribution of soil phosphorus fractions. Soil Sci. 161:436443.

CONCLUSIONS
Incorporation of plant residues into soils can strongly influence POM-P. Changes in this OM pool could be used to describe the initial release of P from plant residues and the microbiological transformations of the substrate during decomposition. Changes in the amount of POM-P indicated a rapid initial release of P from the residues followed by an accumulation of P suggesting a microbial immobilization of P in the decomposing residues. As a result of this P immobilization, and depending on the residue type, up to 30% of the plant P was retranslocated into the decomposing residues during the decomposition process. A correspondence between the increase in POM-P and the fungal colonization of POM-P suggests the possible role of soil fungi in the P immobilization process in decomposing residues, which could be particularly important in acid soils where there is often a predominance of fungal compared with bacterial populations. The amount of P immobilized in the sorghum and crotalaria residues accounted for the observed differences in short-term P availability from these plant residues. However, the mechanisms by which plant factors, other than the C/P ratio, are regulating microbial immobilization of P are not completely understood and further research is needed to assess the long-term effect and the influence of changes in the soil environment derived from agricultural practices. Seasonal variations in climate, soil biological activity, amount of residue input, and competition with plant roots are some of the factors that could potentially modify the immobilization of P in decomposing residues under field conditions.
ACKNOWLEDGMENTS This research was supported by the Humanistic and Scientific Development Council, Consejo de Desarrollo Cientifico y Humanistico, Universidad Central de Venezuela. Project: Soil Nutrient Cycling in dry tropical agroecosystems from Venezuela. We gratefully acknowledge the assistance provided by K. Doxtader and Serita Frey who gave invaluable suggestions for assessing fungal colonization of the POM and provided helpful guidelines in the image analysis procedures. Appreciation is extended to John Chandler (Dep. of Anatomy and Neurobiology, Colorado State University), Dan Reuss (Natural Resource Ecology Laboratory, Colorado State University), and J. Self (Soil Testing Laboratory, Colorado State University) for their assistance in the microbiological and soil chemical analysis. Thanks are also extended to Ana Maria Manzanedo and Amelia Alba for their laboratory assistance.

SALAS ET AL.: PARTICULATE ORGANIC MATTER AND PHOSPHORUS CYCLING

189

Mafongoya, P.L., P. Barak, and J.D. Reed. 2000. Carbon, nitrogen and phosphorus mineralization of tree leaves and manure. Biol. Fertil. Soils 30:298305. Magid, J., A. Gorissen, and K.E. Giller. 1996. In search of the elusive active fraction of soil organic matter: Three size-density fractionation methods for tracing the fate of homogeneously 14C-labelled plant materials. Soil Biol. Biochem. 28:8999. Maroko, J.B., R.J. Buresh, and P.C. Smithson. 1999. Soil phosphorus fractions in unfertilized fallow-maize systems on two tropical soils. Soil Sci. Soc. Am. J. 63:320326. Martin, J.K., and R.B. Cunningham. 1972. Factors controlling the release of phosphorus from decomposing wheat roots. Aust. J. Biol. Sci. 26:715727. McLaughlin, M.J., A.M. Alston, and J.K. Martin. 1988a. Phosphorus cycling in wheat-pasture rotations. I. The source of phosphorus taken up by wheat. Aust. J. Soil Res. 26:323331. McLaughlin, M.J., A.M. Alston, and J.K. Martin. 1988b. Phosphorus cycling in wheat-pasture rotations. II. The role of the microbial biomass in phosphorus cycling. Aust. J. Soil Res. 26:333342. McLaughlin, M.J., A.M. Alston, and J.K. Martin. 1988c. Phosphorus cycling in wheat-pasture rotations. III. Organic phosphorus turnover and phosphorus cycling. Aust. J. Soil Res. 26:342352. McTiernan, K.B., P. Ineson, and P.A. Coward. 1997. Respiration and nutrient release from tree litter mixtures. Oikos 78:527538. Melillo, J.M., and J.D. Aber. 1984. Nutrient immobilization in decaying litter: An example of carbon-nutrient interaction. p.193215. In J. H. Cooley and F. B. Golley (ed.) Trends in ecological research for the 1980s.. Plenum Press, New York. Murphy, J., and J.P. Riley. 1962. A modified single method for the determination of phosphate in natural waters. Anal. Chim. Acta 27:3136. Nziguheba, G., C.A. Palm, R. Buresh, and P. Smithson. 1998. Soil phosphorus fractions and adsorption as affected by organic and inorganic sources. Plant Soil 198:159168. Olsen, S.R., and L.E. Sommers. 1982. Phosphorus. p. 403430. In A. L.Page et al. (ed.) Methods of soil analysis. Part 2. 2nd ed. Agron. Monogr. 9. ASA and SSSA, Madison, WI. Palm, C.A., and P.A. Sanchez. 1991. Nitrogen release from the leaves of some tropical legumes as affected by their lignin and polyphenolic contents. Soil Biol. Biochem. 23: 8388. Parfitt, R. L., K. R. Tate and R. B. McKercher. 1994. Measurement of phosphorus mineralization using an anion exchange membrane. Commun. Soil Sci. Plant Anal. 25(19&20):32093219. Phillips, J.M., and D.S. Hayman. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55:158161.

Salas, A. 2001. Phosphorus cycling during decomposition of plant residues in weathered soils from the tropics: Influence of plant factors. Ph.D. Diss. Colorado State University, Fort Collins, CO. Sakamoto, K., and Y. Oba. 1994. Effect of fungal to bacterial biomass ratio on the relationship between CO2 evolution and total microbial biomass. Biol. Fertil. Soils 17:3944. SAS Institute. 1990. SAS/STAT users guide, vol. 2. Version 6 ed. SAS Institute, Inc. Cary, NC. Schomberg, H.H., and J.L. Steiner. 1999. Nutrient dynamics of crop residues decomposition on a fallow no-till soil surface. Soil Sci. Soc. Am. J. 63:607613. Shang, C., D.E. Caldwell, J.W.B. Stewart, H. Tiessen, and P.M. Huang. 1996. Bioavailability of organic and inorganic phosphates adsorbed on short-range ordered aluminum precipitate. Microb. Ecol. 31:2939. Six, J., E.T. Elliot, K. Paustian, and J.W. Doran. 1998. Aggregation and soil organic matter accumulation in cultivated and native grassland soils. Soil Sci. Soc. Am. J. 62:13671377. Six, J., P.A. Schultz, J.D. Jastrow, and R. Merckx. 1999. Recycling of sodium polytungstate used in soil organic matter studies. Soil Biol. Biochem. 31:11931196. Swift, M.J., O.W. Heal, and J.M. Anderson. 1979. Decomposition in terrestrial ecosystems. Blackwell, Oxford. Tian, G., B.T. Kang, and L. Brussard. 1992. Biological effects of plant residues with contrasting chemical composition under humid tropical conditionsDecomposition and nutrient release. Soil Biol. Biochem. 24:10511060. Umrit, G., and D.K. Friesen. 1994. The effect of CP ratio of plant residues added to soils of contrasting phosphate sorption capacities on P uptake by Panicum maximum (Jaq.). Plant Soil 158:275285. Van Soest, P.J. 1967. Use of detergents in the analysis of fibrous deeds. II. A rapid method for the determination of fiber and lignin. J. AOAC Int. 50:5055. Vanlauwe, B., L. Dendooven, and R. Merckx. 1994. Residue fractionation and decomposition: The significance of the active fraction. Plant Soil 158:263274. Vanlauwe, B., O. Nwoke, N. Sanginga, and R. Merckx. 1996. Impact of residue quality on the C and N mineralization of leaf and root residues of three agroforestry species. Plant Soil 183:221231. Vanlauwe, B., N. Sanginga, and R. Merckx. 1998. Soil organic matter dynamics after addition of nitrogen-15-labeled leucaena and dactyladenia residues. Soil Sci. Soc. Am. J. 62:461466. Vanlauwe, B., K. Aihou, S. Aman, B.K. Tossah, J. Diels, O. Lyasse, S. Hauser, N. Sanginga, and R. Merckx. 2000. Nitrogen and phosphorus uptake by maize as affected by particulate organic matter quality, soil characteristics, and land-use history for soils from the West African moist savanna zone. Biol. Fertil. Soils 30:440449.