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TECHNICAL REPORTS

Application of Two Organic Amendments on Soil Restoration: Effects on the Soil Biological Properties

^a Departamento de Cristalografía, Mineralogía y Química Agrícola, E.U.I.T.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spain
^b Departamento de Conservación de Suelos y Agua y Manejo de Residuos Orgánicos, Centro de Edafología y Biología Aplicada del Segura, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain

^* Corresponding author (mtmoral{at}us.es)

Received for publication December 16, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
One method for recovering degraded soils in semiarid regions is to add organic matter to improve soil characteristics, thereby enhancing biogeochemical nutrient cycling. In this paper, we studied the changes in soil biological properties as a result of adding a crushed cotton gin compost (CCGC) and a poultry manure (PM) for 4 yr to restore a Xerollic Calciorthid located near Seville (Guadalquivir Valley, Andalusia, Spain). Organic wastes were applied at rates of 5, 7.5, and 10 Mg organic matter ha^–1. One year after the assay began, spontaneous vegetation had appeared in the treated plots, particularly in that receiving a high PM and CCGC dose. After 4 yr, the plant cover in these treated plots was around 88 and 79%, respectively, compared with 5% for the control. The effects on soil microbial biomass and six soil enzymatic activities (dehydrogenase, urease, BBA-protease, ß-glucosidase, arylsulfatase, and alkaline phosphatase activities) were ascertained. Both added organic wastes had a positive effect on the biological properties of the soil, although at the end of the experimental period and at high dosage, soil microbial biomass and soil enzyme activities were generally higher in the PM-amended soils compared to the CCGC-amended soils. Enzyme activity from the PM-amended soil was 5, 15, 13, 19, 22, 30, and 6% greater than CCGC-amended soil for soil microbial biomass, urease, BBA-protease, ß-glucosidase, alkaline phosphatase, arylsulfatase, and dehydrogenase activities, respectively. After 4 yr, the percentage of plant cover was >48% in all treated plots and 5% in the control.

Abbreviations: CCGC, crushed cotton gin compost • PM, poultry manure

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
SOIL ORGANIC MATTER, nutrients, and biological activity contribute to ecosystem-level process and are important for productivity, community structure, and fertility in terrestrial ecosystems (Stevenson, 1994). In recent years, the application of organic wastes with a high organic matter content, such as fresh and composted urban wastes (Garcia et al., 1997; Ros et al., 2003) and sewage sludge (Moreno et al., 1999) to semiarid soils has become a common environmental practice for maintaining soil organic matter, reclaiming degraded soils, and supplying plant nutrients. However, the influence of organic matter on soil properties depends on amount, type, and size of added organic materials (Nelson and Oades, 1998; Barzegar et al., 2002). The effect of each organic material on soil properties depends on its dominant component.

Since many enzymes respond immediately to changes in soil fertility status, they can be used as potential indicators of soil quality for sustainable management (Garcia et al., 2000). Measurement of the soil enzymatic activities can be utilized as an indicator of the re-establishment of connections between the biota and restoration of function in degraded systems. Enzymes may react to changes in soil management more quickly than other variables and therefore may be useful as early indicators of biological changes (Bandick and Dick, 1999; Masciandaro et al., 2004). In fact, they may also indicate the soil's potential to sustain microbiological activity (Paul and Clark, 1989).

Oxidoreductase and hydrolase enzymes act on the basic processes of organic matter decomposition. In this respect, dehydrogenase activity is an oxidoreductase enzyme which has been used as a measurement of overall microbial activity (Garcia et al., 1997; Pascual et al., 1998; Masciandaro et al., 2004), since it is an intracellular enzyme related to the oxidative phosphorylation process (Trevors, 1986). Other hydrolytic enzymes involved in the cycling of principal nutrients such as ß-glucosidase, urease, phosphate, and arylsulfatase linked to C, N, P, and S, are sensitive indicators of management-induced changes in soil properties due to their strong relationship with soil organic matter content and quality (Pascual et al., 1998; Masciandaro and Ceccanti, 1999; Masciandaro et al., 2004). These parameters are the most sensitive to the changes which occur in a soil, and provide rapid and accurate information on changes in soil quality, and will help decide the best ways of maintaining sustainable productivity.

The objective of this study was to evaluate the effects of using two different organic wastes (crushed cotton gin compost and poultry manure) as soil amendment at different rates on some soil biological properties (soil microbial biomass and soil enzymatic activities) in a semiarid Mediterranean agroecosystem.

	MATERIALS AND METHODS

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Soil, Organic Wastes, and Climatic Conditions
The study was conducted from October 1999 to October 2003 near Sevilla (Guadalquivir Valley, Andalusia, Spain) on a Xerollic Calciorthid (Soil Survey Staff, 1987). The general properties of this soil (0–25 cm) are shown in Table 1. The organic wastes applied were a crushed cotton gin compost (CCGC) and poultry manure (PM).

The organic wastes were applied to the soil surface at the same amount on 14 Oct. 1999, 17 Oct. 2000, 18 Oct. 2001, and 14 Oct. 2002. The quality of both organic wastes was the same during the experimental period. In this respect, CCGC and PM were kept in refrigeration 0°C after application in the first year so that there were no problems of mineralization of the organic compounds of these products.

Soil samples (0–25 cm) were collected from each plot with a gauge auger (30-mm diameter) on 18 Apr. 2000, 20 Oct. 2000, 15 Apr. 2001, 17 Oct. 2001, 14 Apr. 2002, 11 Oct. 2002, 13 Apr. 2003, and 19 Oct. 2003, respectively. Three subsamples were collected from each plot. After air drying, the soil samples were ground to pass a 2-mm sieve and stored in sealed polyethylene bags at 4°C until analysis.

Analytical Determinations
Plant cover, or percentage of soil covered by the octagonal projection of the aerial part of each plant, was determined by the lineal intercept method (Canfield, 1941).

Soil microbial biomass was determined using the CHCl₃ fumigation–extraction method (Vance et al., 1987). Samples of moist soil (10 g) were used, and K₂SO₄–extractable C was determined using dichromate digestion. Microbial biomass C was calculated (Vance et al., 1987) using the equation: biomass C = 2.64E_C, where E_C = (organic C in K₂SO₄ from fumigated soil) – (organic C in K₂SO₄ from unfumigated soil).

The levels of six enzymatic activities in the soil were measured. Dehydrogenase activity was determined by the method of Garcia et al. (1993). In this procedure, 0.1 g of soil was exposed to 0.2 mL of 4% INT (2-p-iodo-3-nitrophenyl 5-phenyl tetrazolium chloride) in distilled water for 20 h at 22°C in darkness. The iodonitrotetrazaolium formazan (INTF) formed was extracted with 10 mL of a 1:1.5 mixture of ethylene chloride and acetone by shaking vigorously for 2 min. INTF was measured in a spectrophotometer at 490 nm. Controls were prepared without substrate.

Urease activity was determined by the buffered method of Kandeler and Gerber (1988). In this procedure, 0.5 mL of a solution of urea (0.48%) and 4 mL of borate buffer (pH 10) were added to 1 g of soil in hermetically sealed flasks, and then incubated for 2 h at 37°C. The ammonium content of the centrifuged extracts was determined by a modified indophenol-blue reaction. Controls were prepared without substrate to determine the ammonium produced in the absence of added urea.

Protease activity in the form of N--benzoyl-L-argininamide (BBA) protease was measured by a modification of the method proposed by Nannipieri et al. (1980). Phosphate buffer (2 mL, pH 7) and 0.5 mL of substrate (0.03 M N--benzoyl-L-argininamide) were added to 0.5 g of soil. Again, controls were prepared without substrate.

Alkaline phosphatase activity was measured by the method of Tabatabai and Bremner (1969) except that incubation was at 30°C in maleate buffer (2 mL, pH 6.5) for 90 min and 0.5 mL of substrate (0.115 p-nitrophenyl phosphate) added to 0.5 g to soil. Controls were prepared without substrate.

ß-Glucosidase activity was determined using 2 mL of 0.1 M maleate buffer (pH 6.5) and 0.5 mL of 50 mM p-nitrophenyl-ß-D-glucopyranoside (PNG) to 0.5 g of soil. The rest of method was the same as for alkaline phosphatase activity (Masciandaro et al., 1994).

Arylsulfatase activity was determined by the method of Tabatabai and Bremner (1970). Four milliliters of acetate buffer (pH 5.8) and 1 mL of p-nitrophenylsulphate (pNPS) were added to 1 g of soil and then incubated for 1 h at 37°C. One milliliter of 0.5 M CaCl₂ and 10 mL of 0.5 M NaOH were then added. Nitrophenyl was determined in a spectrophotometer at 410 nm. Again, controls were prepared without substrate.

In the laboratory and in the subsamples at the end of the experiment, soil respiration for all treatments was measured by incubation for 3, 7, 15, 30, 45, 60, 90, and 120 d. Total C-CO₂ collected in the NaOH flasks was determined by the addition of an excess of 1.5 M BaCl₂ followed by titration with standardized HCl using a phenolphthalein indicator (Zibilske, 1994).

Statistical Analysis
Analysis of variance (ANOVA) was performed using the Statgraphics Version 5.0 software package (Statistical Graphics Corporation, 1991). ANOVA was based on the LSD criterion (least significant differences between means using Student's t), considering a significance level of P < 0.05 throughout the study. For the ANOVA analysis, the triplicate data were used for each fertilizer treatment and every experimental season, although in the tables the values that appear are the average of the triplicate. The LSD test were employed when the F statistic for the treatment effect was significant and not significant. The LSD values are presented followed by the significance level.

	RESULTS

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Plant Cover
One year after the organic amendment, the treated plots were covered with spontaneous vegetation. The most abundant species were Italian bugloss (Anchusa azurea P. Mill.), common borage (Borago officinalis L.), crowndaisy [Chrysanthemum coronarium (L.) Tzvelev], annual wallrocket [Diplotaxis muralis (L.) DC.], purple mistress [Moricandia arvensis (L.) DC.], and thyme (Thymus hyemalis Lange). Table 4 shows the evolution of percentage of cover plant after the application of CCGC and PM during the experimental period. After 4 yr, the percentage of plant cover decreased in the following order: plot treated with PM at high dose (10 Mg organic matter ha^–1, 88% plant cover) > plot treated with CGCC at high dose (10 Mg organic matter ha^–1, 79% plant cover) > plot treated with PM at medium dose (7.5 Mg organic matter ha^–1, 70% plant cover) > plot treated with CGCC at medium dose (7.5 Mg organic matter ha^–1, 62% plant cover) > plot treated with PM at low dose (7.5 Mg organic matter ha^–1, 55% plant cover) > plot treated with CGCC at low dose (5 Mg organic matter ha^–1, 48% plant cover) > control soil (5% plant cover).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

Microbial Activity in Amended Soil
The supply of readily metabolizable C in the organic wastes is likely to have been the most influential factor contributing to the biomass C increases. According to De Neve and Hofman (2000), Trinsoutrot et al. (2000), and Tejada and Gonzalez (2003, 2004, 2006), soil microbial biomass responds rapidly to additions of readily available C. The positive effect on microbial biomass observed in the soils amended with compost is due to a direct (microbial growth in these composts; Pascual et al., 1998) and indirect effect (improvement of plant growth). These results are in agreement with Ros et al. (2003) who found an increase in soil microbial biomass after the addition to soil of fresh and composted urban waste.

The fact that soil microbial biomass and soil respiration were higher in PM than in CGCC-amended soils may have been due to a greater labile fraction of organic matter in the former product. The labile fraction of organic matter is the most degradable and therefore the most susceptible to mineralization (Cook and Allen, 1992), acting as an immediate energy source for microorganisms. In addition, the nutrient content (N and P) was higher in PM than in CGCC, and these nutrients may have increased the quantity and activity of microorganisms (Marin, 2004).

Curves representing cumulative CO₂–C with time show that the slope at the outset was higher in the soil amended with PM, suggesting that, in this treatment, the carbon substrates are mineralized more rapidly, and that the greater microbial biomass derived from this treatment would have been able to degrade a greater quantity of substrates.

Soil enzymes are biological catalysts of specific reactions and these reactions, in turn, depend on a variety of factors (Burns, 1978) such as the presence or absence of inhibitors, type of amendment, crop type, etc. Soil enzymes are good markers of soil fertility since they are involved in the cycling of the most important nutrients. The incorporation of organic amendments to soil influences soil enzymatic activities because the added material may contain intra- and extracellular enzymes and may also stimulate microbial activity in the soil (Goyal et al., 1993; Pascual et al., 1998).

The oxyreductase enzyme (dehydrogenase activity) has been proposed as a measure of overall microbial activity (Masciandaro et al., 2001), since it is an intracellular enzyme related to oxidative phosphorylation processes (Trevors, 1986). Benefield et al. (1977) and Garcia et al. (1997) found that dehydrogenase activity is a good index of the soil microbial biomass in semiarid Mediterranean areas.

According to Rao and Pathak (1996) and Liang et al. (2005), the incorporation of organic amendments to soil stimulated dehydrogenase activity because the added material may contain intra- and extracellular enzymes and may also stimulate microbial activity in the soil (Pascual et al., 1998; Liang et al., 2005). The microbial populations favored by the root exudates of the plants growing in the plots may also have contributed to the stimulation of dehydrogenase activity. The high level of dehydrogenase activity in the soil treated with PM suggests the availability of a high quantity of biodegradable substrates (which is in agreement with the higher content of labile C observed in these soils) and hence an improvement in their microbial activity.

Measurement of soil hydrolases provides an early indication of changes in soil fertility since they are related to the mineralization of important nutrient elements such as C, N, P, and S (Ceccanti et al., 1994). It is clear that the N cycle was modified in the soils treated with both organic wastes (CGCC and PM). The observed stimulation of urease and BBA-protease activity (related to the N cycle) is appreciable even with high doses of organic amendments, probably due to the higher microbial biomass produced in response. Some studies indicated that high doses of some organic materials can introduce toxic compounds (heavy metals) which could to have a negative effect on enzyme activities (Garcia et al., 1994). Our materials do not have high quantities of heavy metals and consequently, high doses of these materials will not have toxicity of this type. Garcia et al. (1994) indicated that the positive effect of organic amendments on soil biological quality is due to the stimulation of microbial growth and/or to the addition of microbial cells or enzymes with the amendment which can counteract the negative effect produced by some toxic compounds.

Values of urease activity decreased in the control soil with a sparse plant cover (5%). Plant debris remaining in the soil, as well as root exudates, provide nitrogenous substrates (Garcia et al., 2000), which can induce the synthesis of these enzymes in arid soils as occurred in the amended soil (plant cover > 48%). Our results agree with those of Speir et al. (1980) who also found that urease and protease activities of plantless soils gradually decreased over 5 mo, whereas increases were observed in vegetated soils.

ß-Glucosidase catalyzes the hydrolysis of ß-glucosides in soil and is one of the enzymes involved in the decomposition of plants (Hayano and Tubaki, 1985). ß-Glucosidase activity reflects the state of the organic matter and the processes occurring therein (Garcia et al., 1994). This activity was low in the control soil throughout the experiment, probably due to the low soil organic matter content and the resistance to decomposition of this type of organic matter (Garcia et al., 1996). According to Garcia et al. (1997) the vegetation that developed in the experiment in the organic amended soils had a positive influence on the synthesis of this enzyme (Table 7).

In addition, soil arylsulfatase and soil alkaline phosphatase activities were stimulated at high doses of organic amendment. The demand for P by plants and soil microorganisms may have been responsible for the stimulation in the synthesis of this enzyme (Garcia et al., 1994). Moreover, the process involved in the degradation of organic matter may be monitored through hydrolases, such as phosphatase. According to Rao and Tarafdar (1992), increases in phosphatase activity indicate changes in the quantity and quality of soil phosphoryl substrates. The supply of readily metabolizable C in the organic wastes is likely to have been the most influential factor contributing to increased soil arylsulfatase and soil phosphatase activities. However, our results indicated that soil enzymatic activities are slightly higher in PM than CGCC-amended soils. This may be due to the different chemical of the organic wastes added to the soil. The principal difference between both organic wastes was the humic and fulvic acid concentrations. Crushed cotton gin compost has a higher humic acid concentration than PM.

Humic and fulvic acids differ in their structures. Fulvic acids are macromolecules with a lower polymerization index, lower molecular weight, and lower aromaticity and are more highly charged and polar than humic acids (Stevenson, 1994).

Visser (1985) attributed the effect of humic and fulvic acids to inducing a change in microorganism metabolism, allowing the microorganisms to grow on substrates that they previously could not utilize. The lower molecular weight fractions (fulvic acids) appeared to be more effective than the humic acids. This may be due to a greater labile fraction of organic matter of PM than for the CGCC. The labile fraction of organic matter is the most degradable and therefore the most susceptible to mineralization (Cook and Allen, 1992), acting as an immediate energy source for microorganisms. The degradation of added organic matter generates a high demand for N, since the fungi and bacteria that intervene in its transformation have lower C to N ratios than the organic matter that they consume. For this reason, organic matter with greater higher N content is degraded more quickly, favoring microbial growth and soil enzymatic activity (Cotrufo et al., 2000).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The application of CGCC and PM at the doses studied under dry climatic conditions improved the biological properties of the soil. These organic treatments also favor the appearance of spontaneous vegetation, which will protect the soil and will contribute to its restoration. Consequently, the addition of these organic materials may be considered a good strategy for recovering semiarid areas. However, the soil microbial biomass, soil respiration, and soil enzymatic activities were higher in PM- than CGCC-amended soils. This may be due to a greater labile fraction of organic matter in PM than in CCGC and therefore it is most degradable and susceptible to rapid mineralization.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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