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USDA-ARS Integrated Farming and Natural Resour. Res. Unit, 2413 E. Hwy. 83, Weslaco, TX 78596-8344
* Corresponding author (lzibilske{at}weslaco.ars.usda.gov).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Oxygen availability in soil surrounding the residue can differ markedly with the vertical position of the residue, with lower concentrations often dominating at lower soil depths. It has been shown that low soil O2 concentrations slow residue decomposition (Parr and Reuszer, 1959). Qualitative aspects of decomposition are also affected by O2 availability, which can be attributed to well-known differences between aerobic and anaerobic decomposition pathways.
The biochemistry of the residues also influences decomposition dynamics. Plant polyphenolic content has been shown to affect C and N transformations in soil (Palm and Sanchez, 1990). A high content of polyphenolic compounds in residues extends C residence time in soils (Tian et al., 1993). Accordingly, using crop residues high in polyphenolic content may promote C retention (Tian and Brussard, 1997), ameliorating, to some extent, the more rapid soil organic matter losses in tropical climates (Jenkinson and Anayaba, 1977; Sanchez and Logan, 1992; Shang and Tiessen, 1998). Polyphenolics have been shown to be important in soil N dynamics and other biologically mediated processes such as aggregate formation and stabilization (Martens 2000). Little information on the role of polyphenolics on soil nutrient cycles is available (Hättenschwiler and Vitousek, 2000).
For buried residues, including roots, O2 availability is a strong regulator of decomposition. Molecular O is required for polyphenol oxidase (Freeman et al., 2001; 2004; Fenner et al., 2005), which is an important enzyme in the processes of lignin and polyphenolic decomposition and contributes to the "enzymic latch" concept in which phenol oxidase and other hydrolytic enzymes are inhibited by low O2 levels, slowing C mineralization (Freeman et al., 2001). These reports document the inhibition of phenol oxidases by anaerobic conditions, but data that quantify the relationship between O2 concentration and oxidase activity are lacking. Conditions that restrict O2 diffusion in the soil should restrict polyphenol oxidase activity, allowing the accumulation of polyphenolics, which have been shown to decrease other enzyme activities (Aerts et al., 1999). This is an important proposition that embraces the role of reduced tillage (i.e., lower soil O2), in combination with cover crop management (i.e., possibly higher content of polyphenolics), in establishing conditions that promote the accumulation of soil organic matter in hot climates.
The purpose of this experiment was to determine the effects of O2 availability on residue polyphenolic compounds, soil phenol oxidase activity, peroxidase activity, arginine ammonification (as an index of N mineralization), and water-extractable C (as an index of mobile soil C) in soil amended with contrasting crop residues.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Plant Materials
Fully developed green stems and leaves of field-grown cowpea and grain sorghum were collected from field plots within 20 m of the location of soil collection, described below. The samples were oven dried (45°C), ground to pass a no. 40 sieve (
0.5-mm openings), and mixed thoroughly. Plant C and N were determined by dry combustion (Elementar VarioMax CN analyzer, Elementar Americas, Mt. Laurel, NJ) and found to contain 40.4% C and 3.27% N (cowpea) and 41.5% C and 2.2% N (sorghum). Samples of ground plant tissue were extracted three times with 20 mL of 50% (v/v) aqueous methanol at 75°C for 1 h (Osono and Takeda 1999) to determine the initial polyphenolic content in the tissues. The tissues were found to contain 5.56 and 8.91 g kg–1 tissue total phenolics for cowpea and sorghum, respectively. These test crops were chosen because they are used as cover crops in this region for improving soil organic matter and contribute large amounts of plant matter to the soil during preparations for the next crop.
Soil Preparation
Soil was collected by sampling probe from the surface 20 cm of a fallow Delfina fine sandy loam (fine-loamy, mixed, hyperthermic Aquic Paleustalf) in an agricultural field at the plant sample collection site, near Monte Alto, TX, in a semiarid subtropical climatic zone (26°26' N, 97°58' W; elevation 13 m; mean air temperature during the growing season 29°C). Soil was mixed field moist, crushed gently to pass a 2-mm sieve, and stored in plastic bags at 15°C until used. The soil collection site had been in commercial grain sorghum production for 14 yr. Selected soil properties are: soil particle size (following dispersion): sand, 80%; silt, 12%; clay, 8%; pH 6.9 (glass electrode, 1:2 soil/water); total organic C, 3.15 g kg–1; total organic N, 0.32 g kg–1 (both by dry combustion); P (strong Bray), 64 mg kg–1; cation exchange capacity (summation of bases), 5.5 cmol kg–1.
Experimental Units
Soil and ground plant material were mixed thoroughly by hand at the rate of 0.5 g dry plant material to 10 g soil (dry equivalent). This relatively large amount of residue was used to simulate a surface soil that becomes highly enriched with organic materials when tillage is reduced. In addition, high amendment rates were used to ensure that polyphenolic concentrations would be sufficient to observe changes during a long incubation to properly test the hypotheses. Soil moisture was adjusted to 65% of water-holding capacity. The soil samples were transferred to 7 by 7 cm bags sewn from nylon mesh (1.0-mm openings). The thickness of the bags containing soil was not >1 cm. Bags were closed with a paper clip and replicates were placed onto a wire rack configured to fit into a 2-L mason jar lying on its side. Wire racks were used to facilitate gas exchange between the enclosed atmosphere and the soil in the bags. A wetted paper towel was placed into the jars beneath the racks to maintain humidity. Towels were rewetted as necessary. Jars were sealed with lids fitted with two gas sampling septa. A hypodermic needle was inserted into one of the septa to serve as a gas outlet. Flushing was accomplished by introducing flowing N2 from a pressurized N2 source through tubing connected to a hypodermic needle inserted into the second septum. The N2 flow was periodically interrupted to determine O2 concentration achieved in the headspace. Gas samples were taken by syringe and needle and analyzed by gas chromatography (Zibilske, 1994). In this way, O2 concentration in three replicate jars was adjusted to 0.5 or 10%. The 21% O2 treatment was established by sealing jars and flushing with room air. Jars were allowed to equilibrate for 3 h at room temperature and resampled for O2 content. If needed, jars were flushed with N2 or room air to reestablish the original O2 concentrations. Jars were checked daily for 10 d and biweekly afterward for headspace O2 content. If O2 concentrations deviated 
5% of nominal, jars were flushed to reestablish the desired O2 concentrations. Vessels were incubated in darkness at 30°C, which is a commonly observed soil temperature in the spring and early summer at this latitude. At Time 0 and on Days 7, 14, 28, 42, 70, and 110, jars were opened, replicate bags were removed and the contents subjected to the biochemical assays described below.
Biochemical Analyses
Phenol oxidase activity was determined as described by Matocha et al. (2004) and the 96-well microtiter plate procedures of Sinsabaugh et al. (2005) and Larson et al. (2002), measuring the absorption increase after 4 h at 450 nm with the oxidation of L-3,4-dyhydroxyphenylalanine (L-DOPA) to 2-carboxy-2,3-dihydroindole-5,6-quinone. Absorbances were determined with a Dynatech MR5000 microplate reader (Dynex Technologies, Chantilly, VA). After correction for controls, absorbance values were converted to product concentrations per unit of time and soil mass using the molar absorption coefficient as described by Matocha et al. (2004).
Peroxidase activity was determined by adding 200 µL of 0.2 mM H2O2 to the reaction mix for L-DOPA oxidation described above. Absorbances above those obtained with the L-DOPA alone were interpreted to result from peroxidase activity (Bending and Read, 1996). Absorbance values reported are the differences between the absorbance resulting from the phenol oxidase assay alone and that including peroxide and have been corrected for controls.
Water-extractable polyphenolic compounds were determined in the extracts by colorimetric reaction with Folin–Denis reagent (King and Heath 1967; Mueller-Harvey, 2001) using an aqueous tannic acid standard line.
The term water-extractable C is used instead of dissolved organic matter since samples probably contained colloidal C as well as dissolved C; therefore, WEC may be a more accurate description. Water-extractable C was determined by mechanically shaking 5 g soil in 50 mL deionized water in a conical centrifuge tube for 30 min. Tubes were centrifuged at 250 x g, membrane filtered (0.45 µm), and the liquid was either analyzed immediately or frozen (–20°C) until analysis. The extracts were analyzed for total organic C with a Dohrmann DC-190 Total Organic Carbon Analyzer (Rosemount Analytical, Santa Clara, CA).
Arginine ammonification was determined by the Bonde et al. (2001) modification of the indophenol blue method. The method was modified by incubating the assay at 30°C, a common soil temperature at this latitude, instead of 20°C. Since O2 concentration was a variable in these experiments, arginine ammonification was chosen as an index of N mineralization to minimize the confounding effect that nitrification might cause if extractable inorganic N had been used as the index. It is still possible, however, that some nitrification of released NH4+ could have occurred in the samples because they were incubated under aerobic conditions during the 1-h assay. This effect was assumed to be negligible.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The 21% and 10 O2 treatments with cowpea amendment showed very slow increases from Day 0 to 28 (Fig. 1, upper), but more rapid increases in PPO activity were observed with sorghum amendment (Fig. 1, lower). In contrast to cowpea amendments, sorghum residues caused peaks in PPO activities around 28 d, while cowpea residues elicited slow increases for the entire incubation period. The differences in PPO activity patterns between the two residues may be explainable by differences in N content between the residues, or perhaps due to other substrate differences. If this is correct, the sorghum residue may contain more readily available substrate for PPO activity than cowpea, but it also contains less soluble N. Both properties would explain the earlier onset of PPO activity and higher rates of activity in the sorghum treatments. The readily available sources were perhaps exhausted more rapidly in the sorghum treatment. The cowpea contained less total phenolics than the sorghum at the outset, 5.56 vs. 8.91 g kg–1 tissue, so a difference in availability of the substrates between the two could explain a higher activity with the sorghum amendment in that period.
Peroxidase Activity
Another common phenol oxidase is peroxidase. Both peroxidase and phenol oxidase are simultaneously active and prepare lignin-like molecules for ring fission, a necessary step in the mineralization of lignin materials. Peroxidase activities in the cowpea- and sorghum-amended soils were generally similar during the 110-d incubation. For cowpea (Fig. 4
, upper), there was an increase in activity from Days 0 to 28, but no significant difference appeared until Day 28, when the 21 and 10% O2 treatments were higher than the 0.5% treatment. Between 42 and 70 d, activity in the 21% treatment peaked significantly higher than the 10% treatment, while the 10% treatment remained significantly higher that the 0.5% treatment but essentially changed little for the remainder of the incubation. Decreases in activity were observed in both the 21 and 0.5% O2 treatments after 42 d. By Day 70, the 21 and 10% treatments were similar again, but both were significantly (P = 0.020) higher than the 0.5% treatment after 42 d of incubation.
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It is noteworthy that PO activity increased in the first 28 d, approximately doubling (Fig. 4). This is similar to the change in PPO activity (Fig. 1) in that period, although PO activity was about 10 times greater than PPO activity. This suggests similar controls on PO and PPO activities in this system.
Both PPO and PO activities peaked in the first half of the incubation, and correspond to the rapid decreases in WEP in that period (Fig. 5 ). It should be noted, however, that the inverse relationship does not appear to be applicable to the 0.5% O2 treatment, which caused higher levels of WEP to be maintained, but which correspond to only slightly increased PPO activity but rather higher PO activities in that period. These results suggest an important role for O2 presence in removing polyphenolics from soil solution.
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Sorghum residues (Fig. 2, lower) displayed patterns similar to the cowpea residues. Sorghum started out with a lower content of WEC, but the general patterns of decline were very similar to that of the cowpea in response to O2 treatment. The 0.5% O2 treatment was significantly higher (P < 0.001) than both the 10 and 21% treatments. In contrast to the cowpea, sorghum residues at the 10 and 21% O2 treatments were not significantly different at 42 d of incubation and after.
Dissolved organic C was found to be highly correlated to C mineralization rates (Zsolnay and Görlitz, 1994), but in a low-O2 environment, WEC levels may indicate accumulation of depolymerized organic C and fermentation products and thus may represent an incomplete decomposition of residues and a lesser degree of C mineralization.
Water Extractable Polyphenolics
Polyphenolics have multifaceted effects in soil systems. Effects include slowing organic matter decomposition (Zibilske and Materon, 2005) and slowing N mineralization (Tian et al., 2001), and have been purported to affect plant succession (Schimel et al., 1996).
Water-extractable polyphenolics determined during the incubation are shown in Fig. 5. The 0.5% O2 treatment showed the slowest decline for cowpea (Fig. 5, upper) as well as for sorghum (Fig. 5, lower). Both the 10 and 20% O2 treatments promoted more rapid declines (Fig. 5, upper) and were significantly (P < 0.001) lower than the 0.5% treatment by 7 d of incubation. Water-extractable polyphenolics had essentially been eliminated from the soil by Day 72 in the 10 and 21% treatments. Similar declines in WEP for sorghum were observed (Fig. 5, lower), as the 10 and 21% treatments showed virtually no differences in WEP beyond 28 d.
Persistence of polyphenolics in the 0.5% O2 treatment is probably accounted for by the fact that polyphenol oxidase activity requires molecular O. Freeman et al. (2004) reported that an O2 constraint on polyphenol oxidase activity slows organic matter decomposition. Our results support that assertion and suggest that, due to the similarity of the responses of the 10 and 21% O2 treatments, a substantial reduction in ambient O2 concentration is necessary to effect such a decrease in polyphenol oxidation for both cowpea and sorghum tissues in soil.
Arginine Ammonification
Polyphenol effects on N mineralization can lead to immobilization (Tian et al., 2001), which may tend to slow organic matter mineralization. It would be expected that the presence of soluble polyphenols would slow ammonification, an early step in N mineralization in soils. A negative relationship has been demonstrated between dissolved inorganic N and polyphenol oxidase activity in soil (Matocha et al., 2004). When soils were amended with cowpea residues containing 3.27% N (Fig. 3 upper), ammonification of arginine was significantly (P < 0.001) increased during the course of the incubation in the order 0.5 > 10 > 21%. This is in opposition to expectation. Oxygen presence is not usually considered to be necessary for ammonification of amino N.
Sorghum residue elicited a different pattern for arginine ammonification activity (Fig. 3, lower). Rates did not increase as rapidly as for the cowpea treatment during the first 20 d. Between 14 and 28 d, ammonification rates increased greatly in all O2 treatments, peaking between 28 and 42 d. Similar rates of decline were observed after 42 d, although the 21% O2 treatment was significantly (P < 0.001) lower than both the 0.5 and 10% treatments by 70 d and after. The declines in ammonification rates roughly mirror declines in WEC during the preceding period from Days 0 to 28 (Fig. 2, lower), suggesting a reduction in general microbial activity reflected in diminished levels of WEC. Similar declines in that period were observed for WEP (Fig. 5, lower), suggesting that oxidative losses of WEP followed the pattern seen for WEC. To some degree, these concomitant losses (WEC and WEP) are to be expected during decomposition of soluble C sources in the soil.
Perhaps a minimum amount of soil organic C (SOC), and therefore of soil polyphenolic compounds, is necessary to show marked effects on N mineralization. Extending this concept, if residues containing very high levels of polyphenolics are added to the soil, the level of polyphenolics in the system may attain the level necessary to effect a reduction in N mineralization rates, even though native SOC alone might be insufficient to generate or maintain polyphenolic concentrations high enough to reduce N mineralization. Studies by Palm and Sanchez (1990) and Tian et al. (1993) indicated that SOC can accumulate when tropical legume tree litter is used to amend agricultural soils. Our results suggest that the same might be possible using more temperate plant residues.
Since a relatively large amount of readily decomposable residues was added at the beginning, the rapid loss of WEC in the system and treatment differences in arginine ammonification rates suggest that it is still useful as an indicator of N mineralization.
Synthesis
The overall effects of O2 treatment and residue type on experimental parameters were examined with Pearson correlations (Table 1). Significant negative correlations were determined for combinations of PPO and WEC and also PPO and WEP for both cowpea and sorghum treatments at the 10 and 21% O2 levels. This suggests that PPO was responsible for removing those species from solution. This might be expected, since ready access to those substrates would accelerate their decomposition. The strongest correlations were noted for the 10 and 21% O2 treatments. This again shows the strong inhibitory effect of low O2 availability on those enzyme activities. The assay for PPO and PO involves an aerobic incubation for 4 h. That amount of time may allow for some de novo synthesis in those samples coming from the 0.5 and 10% O2 treatments, but the effect was assumed to be negligible. Significant positive correlations were determined for most WEC and WEP pairings, except for cowpea at 21% O2 and sorghum in 0.5% O2. Apart from the probable confounding among the significant correlations, the nonsignificant pairings suggest that WEC and WEP levels vary independently in those treatment combinations. The reasons for this are unknown, but point to differences in the rates of transformation due to very low O2 content, very high O2 content, and residue quality.
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Comparisons of WEP x ARG pairings reveal significant negative coefficients for several O2 and residue treatments. The strongest relationships were noted for the 10% O2 treatment for both residues, but correlations were strong as well for 0.5% O2 with cowpea and 21% O2 with sorghum. As seen for ARG x WEC pairings, all WEP x ARG comparisons produced negative coefficients. This indicates that the general relationship for this pairing is an inverse one, supporting the proposal that polyphenols slow N mineralization.
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication April 24, 2006.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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  D. C. Olk Improved Analytical Techniques for Carbohydrates, Amino Compounds, and Phenols: Tools for Understanding Soil Processes Soil Sci. Soc. Am. J., October 30, 2008; 72(6): 1672 - 1682. [Abstract] [Full Text] [PDF]
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