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Cover crop and tillage effects on soil enzyme activities following tomato
Soil & Tillage Research 105 (2009) 269–274
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Cover crop and tillage effects on soil enzyme activities following tomato Said A. Hamido, K. Kpomblekou-A * Department of Agricultural and Environmental Sciences, Tuskegee University, 213 Milbank Hall, Tuskegee, AL 36088, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2009 Received in revised form 4 September 2009 Accepted 12 September 2009
Increasing numbers of vegetable growers are adopting conservation tillage practices and including cover crops into crop rotations. The practice helps to increase or maintain an adequate level of soil organic matter and improves vegetable yields. The effects of the practices, however, on enzyme activities in southeastern soils of the United States have not been well documented. Thus, the objectives of the study were to investigate the effects of cover crops and two tillage systems on soil enzyme activity profiles following tomato and to establish relationships between enzyme activities and soil organic carbon (C) and nitrogen (N). The cover crops planted late in fall 2005 included black oat (Avena strigosa), crimson clover (Trifolium incarnatum L.), or crimson clover–black oat mixed. A weed control (no cover crop) was also included. Early in spring 2006, the plots were disk plowed and incorporated into soil (conventional tillage) or mowed and left on the soil surface (no-till). Broiler litter as source of N fertilizer was applied at a rate of 4.6 Mg ha 1, triple super phosphate at 79.0 kg P ha 1, and potassium chloride at 100 kg K ha 1 were also applied according to soil testing recommendations. Tomato seedlings were transplanted and grown for 60 days on a Marvyn sandy loam soil (fine-loamy, kaolinitic, thermic Typic Kanhapludults). Ninety-six core soil samples were collected at incremental depths (0–5, 5–10, and 10–15 cm) and passed through a 2-mm sieve and kept moist to study arylamidase (EC 3.4.11.2), L-asparaginase (EC 3.5.1.1), L-glutaminase (EC 3.5.1.2), and urease (EC 3.5.1.5) activities. Tillage systems affected only L-glutaminase activity in soil while cover crops affected activities of all the enzymes studied with the exception of urease. The research clearly demonstrated that in till and no-till systems, L-asparaginase activity is greater (P 0.05) in plots preceded by crimson clover than in those preceded by black oat or their mixture. Activity of the enzyme decreased from 11.7 mg NH4+–N kg 1 2 h 1 at 0–5 cm depth to 8.73 mg NH4+–N kg 1 2 h 1 at 5–10 cm and 10–15 cm depths in the no-till crimson clover plots. Arylamidase activity significantly correlated with soil organic C (r = 0.699**) and soil organic N (r = 0.764***). Amidohydrolases activities significantly correlated with soil organic N but only urease significantly correlated with soil organic C (r = 0.481*). These results indicated that incorporation of cover crops into rotations may increase enzyme activities in soils. ß 2009 Published by Elsevier B.V.
Keywords: Enzyme activities Conventional tillage No-till Cover crops Broiler litter
1. Introduction Soils are inhabited by a vast array of microbes responsible for breakdown of organic matter and solubilization of nutrients. These microbes are major source of soil enzymes and their dynamics in soils seemed to be related to management practices. Enzymes play an important role in the cycling of nutrients in nature and because soil enzyme activity is sensitive to agricultural practices, it can be used as an index of soil microbial activity and fertility (Benitez et al., 2000). Thus, they are involved in soil mineralization processes (Tate, 1987) and have been related to other soil biological properties (Frankenberger and Dick, 1983). Enzyme activity profiles reflect an essential part of soil functional diversity,
* Corresponding author. Tel.: +1 334 724 4521; fax: +1 334 724 4529. E-mail address: [email protected] (K. Kpomblekou-A). 0167-1987/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.still.2009.09.007
which is controlled by genetic diversity of soil microorganisms, plants and soil animals in close relation to environmental effects and ecological interactions (Nannipieri et al., 2002). Enzymes catalyze a complex web of chemical reactions necessary for decomposition of organic residues, cycling of nutrients, formation of organic matter, and soil structure (Dick, 1994). Amino acid arylamidase and several amidohydrolases have been identified in soils (Acosta-Martı´nez and Tabatabai, 2001). The most important of the enzymes that play significant role in N mineralization are arylamidase, L-asparaginase, L-glutaminase, amidase, and urease. Because enzymes provide a useful tool for long-term monitoring of changes in soil health and quality, they have been proposed as soil fertility indicators (Frankenberger and Dick, 1983). Arylamidase (EC 3.4.11.2) is an enzyme that catalyzes the hydrolysis of an N-terminal amino acid from peptides amides using L-leucine as the amino acid moiety and its activity was detected in soils (Acosta-Martı´nez and Tabatabai, 2000a,b). This
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enzyme catalyzes one of the most important reactions in N mineralization. It releases amino acids from soil organic matter that are used as substrates for amidohydrolases. Previous studies demonstrated that the activity of arylamidase is significantly affected by crop residue managements (Deng and Tabatabai, 1996a,b, 1997) and tillage systems (Acosta-Martı´nez and Tabatabai, 2001). Acosta-Martı´nez and Tabatabai (2001) compared enzyme activities in soil samples collected from no-till/double mulch and tillage with residue management treatment plots. The authors reported the greatest enzyme activity in the no-till/double mulch plot. Amidohydrolases are widely distributed in nature and have been detected in plants, animals, and microorganisms (Tabatabai, 1994). Soil samples (0–15 cm) obtained from Northeast Research Center in Nashua (Iowa) for a period of seven years (1987–1994) demonstrated that liming increased activities of 13 enzymes (Acosta-Martı´nez and Tabatabai, 2000a). Previous studies on the effect of tillage systems and residue management showed that activities of L-asparaginase, L-glutaminase, and urease were generally greater under no-till systems than under conventional tillage systems (Deng and Tabatabai, 1996a,b, 1997). Activities of phosphatases (acid phosphatase, alkaline phosphatase, phosphodiesterase, and inorganic pyrophosphatase) and arylsulfatase were investigated in three tillage systems (no-till, chisel plow, and moldboard plow) in combination with corn residue placements. Activities of the enzymes in no-till/double mulch were significantly greater than those of other treatments studied (Deng and Tabatabai, 1996a). The activities decreased with increasing soil depths and the decreases were associated with a decrease in organic carbon (C) content. Because soil enzyme activities respond to external organic matter inputs and tillage systems, it is important to investigate activities of enzymes directly responsible for nitrogen (N) transformations in soils when legumes and grasses are added to soil under different tillage systems. Thus, understanding profile distribution of these enzymes in soil will be a valuable tool in evaluating the effect of cover crops on N dynamics in soils. The objectives of the present study were to: (i) evaluate under a conventional (disk plow) and a no-till system, soil enzymes activities in tomato plots preceded by crimson clover, black oat, or their mixture and (ii) establish relationships between enzyme activities and soil organic N and/or organic C. 2. Materials and methods The experiment was initiated in fall 2005 at the George Washington Carver Agricultural Experiment Station at Tuskegee University, Tuskegee, AL, on a Marvyn sandy loam (fine-loamy, kaolinitic, thermic Typic Kanhapludults) soil. Prior to establishment of the experiment, the site was in fallow (mainly grasses) for several years. Composite soil samples were collected from 0 to 15 cm depth for soil characterization and fertilizer recommendations. 2.1. Soil analysis and trial description Soil pH (6.1) was determined by a glass electrode (soil:water 1:2.5) and soil cation exchange capacity (<4.6 cmolc kg 1) was determined by using a neutral 1 M NH4OAc solution as described by Chapman (1965). Extractable P (10.0 mg kg 1) was determined as outlined by Olsen and Sommers (1982), while extractable K (78.0 mg kg 1), Mg (73.0 mg kg 1), and Ca (350 mg kg 1) were measured as described by Mehlich (1978). Extractable metals in the solution were determined by using Inductively Coupled Argon Plasma (ICAP) in a Thermo Jarrell Ash, Model 9000 (Jarrel-Ash Division, 1982). Ammonium–N and nitrate (NO3 + NO2 )–N contents of the soil were extracted with 2 M KCl solution and
determined by steam distillation (Bremner and Keeney, 1966) and averaged 2.14 and 1.65 mg kg 1 soil, respectively. The cover crops tested were crimson clover (Trifolium incarnatum L.), black oat (Avena strigosa), crimson clover–black oat mixed, and a weed–control plot without cover crop. The cover crops were planted late in fall 2005, cut and incorporated into soil by disk plow (conventional tillage) or left on the soil surface as a mulch (no-till) late in the following spring. Broiler litter at a rate of 4.6 Mg ha 1, triple super phosphate at 79.0 kg P ha 1, and potassium chloride at 100 kg K ha 1 were applied to all treatments. Tomato (Lycopersicon esculentum Mill.) seedlings were transplanted and grown for 60 days. Following tomato harvest, ninety-six core soil samples (2.5 cm, ID) were collected at incremental depths (0–5, 5–10, and 10– 15 cm) using an auger to investigate arylamidase and amidohydrolases activities in each experimental unit (3 5 m). The experimental design was a split plot with four replications. The four cover crops were randomly assigned to the horizontal strips and the two tillage systems (conventional tillage or no-till) were randomly assigned to the vertical strips. 2.2. Enzyme activities The soil samples were passed through a 2-mm sieve and kept moist in a refrigerator at 4 8C until used. Arylamidase activity was assayed by incubating 1 g moist soil sample with 3 mL of 0.1 M THAM buffer (pH 8.0) and 1 mL 8.0 mM solution of L-leucine bnaphthylamide hydrochloride (Acosta-Martı´nez and Tabatabai, 2000b). The product of the reaction b-naphthylamine was measured at 540 nm (Hiwada et al., 1977) using a Beckman Coulter DU 640 spectrophotometer (Beckman Instruments, Inc. Fullerton, CA). Controls were included as described for the assay with the exception that the substrate L-leucine b-naphthylamide hydrochloride was added after incubation. Amidohydrolases (L-asparaginase, L-glutaminase, and urease) activities were assayed by incubating 5 g moist soil sample with 1 mL 0.5 M solution of L-asparagine, L-glutamine, or 0.2 M solution of urea and 9 mL of THAM [Tris(hydroxymethyl)aminomethane] buffer in the presence of 0.2 mL of toluene (Tabatabai, 1994). The ammonia formed following hydrolysis of L-asparaginase, Lglutaminase, or urease was measured by steam distillation using 20 mL aliquot of the soil suspension and 0.2 g magnesium oxide (MgO) as described by Bremner and Keeney (1966). Appropriate controls were included in all the assays to account for NH4+–N not derived from L-asparagine, L-glutamine, or urea through their respective enzyme activity (Tabatabai, 1994). All results reported are averages of duplicate assays expressed on moisture-free basis. Moisture was determined from loss in weight following drying at 105 8C for 48 h. 2.3. Cover crop biomass Before cutting the cover crops, four areas of 1 m2 were randomly marked on each experimental unit. The plant material within each marked area was cut above-ground and weighed to estimate the mean cover crop biomass of each treatment. A subsample was taken in a brown bag, weighed, transported to the laboratory, and placed in an oven at 65 8C for 72 h to determine the dry-matter yield while the remaining above-ground plant samples were returned to each corresponding plot. 2.4. Statistical analysis Because measures obtained at the three depths are not independent, data were analyzed using MIXED procedure of SAS (SAS ver. 9.1, Institute, Cary, NC, USA) with a repeated measure
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statement that assumed unstructured (co) variances. Denominator degrees of freedom were calculated using the Kenward–Roger option. Means were compared with Fisher’s protected LSD. The SLICE option and individual degree of freedom contrasts were used to explain significant interactions. The level of significance was set at 0.05. 3. Results and discussion In general, cover crops and soil sampling depths significantly (P 0.05) affected arylamidase, L-asparaginase, and L-glutaminase activities in soil. Urease activity was not significantly affected by the presence of cover crops, tillage systems and soil sampling depths. The two-way interactions: cover crop tillage system and tillage system soil sampling depth were the only combination of factors that significantly affected urease activity in soil. The interaction tillage system soil sampling depth was also significant for L-glutaminase activity while the three-way interaction significantly affected only L-asparaginase and L-glutaminase activities. The above-ground cover crops biomass dry-matter yields incorporated in the soils are shown in Table 1. There were no significant differences (P 0.05) among the cover crops tested in term of dry-matter yields produced but the enzyme activities induced by the cover crops varied significantly. 3.1. Effects of cover crops and tillage systems on arylamidase activity Statistical analyses suggested that cover crops and soil sampling depths affected significantly (P 0.05) arylamidase activity (Fig. 1). The average of arylamidase activity in the notill plots was 7.94 mg b-naphthylamine kg 1 h 1 and 7.34 mg bnaphthylamine kg 1 h 1 on the tilled plots. Across all cover crops, the activities were 7.36 and 8.67 mg b-naphthylamine kg 1 h 1 at 0–5 cm depth, 7.81 and 8.38 mg b-naphthylamine kg 1 h 1 at 5– 10 cm depth, and 6.84 and 6.77 mg b-naphthylamine kg 1 h 1 at 10–15 cm depth in the tilled and no-tilled treatments, respectively. Tillage practices by themselves did not significantly affect arylamidase activity in the soil. Fig. 1 showed that at 0–5 cm soil depth, arylamidase activity in the tilled plots was significantly (P 0.05) higher (10.9 mg bnaphthylamine kg 1 h 1) in the black oat plot than in the weed plot (3.13 mg b-naphthylamine kg 1 h 1) or the crimson clover– black oat mixed (7.43 mg b-naphthylamine kg 1 h 1). At 5–10 cm depth, there was no significant difference (P 0.05) between arylamidase activity observed under black oat, crimson clover, or their mixed treatment plots in the conventional tillage plots. However, the three treatments showed higher arylamidase activity than the weed control tilled plot. In addition, at 10–15 cm depth, there was no significant difference among the black oat, the crimson clover, and the mixed plots; the activity of arylamidase was however, greater in those plots than in the control tilled weed plots where the activity was only 3.38 mg b-naphthylamine kg 1 h 1. The activity of arylamidase increased in the tilled plot under crimson clover from 0–5 to 5–10 cm depth but decreased at 10–15 cm. The activity decreased with depths in plots preceded by black oat. Under the crimson clover–black oat mixed tilled plot, the enzyme activity remained constant (7.5 mg b-naphthylamine
Fig. 1. Effect of cover crops on arylamidase activity at different depths in conventional tillage and no-till systems.
kg 1 h 1), the weed control plots also showed constant activities (3.3 mg b-naphthylamine kg 1 h 1) with depths. This suggests that the cover crops increased activity of arylamidase in soil and that microbial respiration was likely higher in the upper plow layer of the soil where black oat was incorporated. Generally, enzyme activities tend to decrease with depths and differentiation among treatments are found often only in the 0–5 cm depth (Ekenler and Tabatabai, 2004; Green et al., 2007). In a Brazilian Cerrado Oxisol, arylamidase and acid phosphatase activities were 18–186% greater under no-till than under disk plow in 0–5 cm depth (Green et al., 2007). However, in a no-till soil, Ekenler and Tabatabai (2004) reported an increase in arylamidase activity from 16.1 at 0–5 cm depth to 19.8 mg b-naphthylamine kg 1 h 1 at 0–15 cm depth. Our results clearly suggested that arylamidase activity is influenced by the presence of crimson clover or black oat and therefore, could be used to monitor soil health and quality under the conditions of this experiment as suggested by previous studies (Dick, 1994). In the no-till plots, arylamidase activity was consistently higher under black oat at 5–10 and 10–15 cm soil depths than under crimson clover or mixed with crimson clover (Fig. 1). The activities under the black oat treatments were 12.6, 12.6, and 10.5 mg bnaphthylamine kg 1 h 1 at 0–5, 5–10, and 10–15 cm, respectively and were significantly (P 0.05) greater than those under crimson clover and the mixture at 10–15 cm depth. Although the activities of arylamidase under black oat remained high at all soil depths, those under crimson clover decreased steadily with depths to 7.18 mg b-naphthylamine kg 1 h 1 at 10–15. Black oat mulch on top of the soil induced arylamidase activity in the top 0–10 cm but the activity was reduced at 10–15 cm depth although not significantly. A slow decomposition of this mulch increased microbial activity contributing to a higher amount of endoenzymes in the viable microbial populations in the topsoil. It appeared like crimson clover significantly depressed activity of arylamidase in the mixture at all depths. Green et al. (2007) reported greater activity of arylamidase under no-till and disk harrow than under
Table 1 Cover crop biomass yields. Cover crop
Fresh yield (Mg ha
Weed Crimson clover Black oat Crimson clover–black oat mixture
2.78 3.35 3.59 5.20
1
)
b ab ab a
Values in the same column followed by the same letter are not significantly different at P 0.05.
Moisture content (%)
Dry-matter yield (Mg ha
76.9 62.9 57.4 59.2
0.64 1.24 1.53 2.12
b ab a a
1
)
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Fig. 2. Effect of cover crops on L-asparaginase activity at different depths in conventional tillage and no-till systems.
Fig. 3. Effect of cover crops on L-glutaminase activity at different depths in conventional tillage and no-till systems.
disk plow. Studies by Ekenler and Tabatabai (2004) also demonstrated greater arylamidase activity in no-till plots than in ridgetill and chisel plow plots at Northwest Research Center site at Sutherland (Iowa, U.S.A.). Our data support previous works and demonstrated that in no-till systems, enzyme activities tend to be stratified in soil profiles and implied that in some cases, to detect enzyme activities, samples must be taken incrementally to avoid a dilution effect.
weed control plots that showed higher activity of the enzyme. In no-till plots, the enzyme activity did not improve even in the presence of crimson clover in the mixed plots. It was evident that black oat significantly depressed L-asparaginase activity in no-till soils. One might suggest that black oat, because of its allelopathic properties, released substances that inhibited activity of the enzyme.
3.2. Effects of cover crops and tillage systems on amidohydrolases activity Tillage practices have profound influences on amidohydrolases activity in soils. Deng and Tabatabai (1996a) showed that activities of amidohydrolases were generally greater in mulch-treated plots than in non-treated plots and were related to soil organic C. In the present studies, activities of the enzymes varied with cover crops and soil depths. 3.2.1. Effects on L-asparaginase activity In general, tillage practices did not have a significant effect on Lasparaginase activity; however, its two-way interaction with cover crops or with soil sampling depths, and three-way interaction were significant. L-Asparaginase activity in plots preceded by crimson clover was higher than that of plots preceded by black oat at all depths and tillage systems (Fig. 2). Under conventional tillage, the activity of L-asparaginase was as low as 0.75 mg NH4+–N kg 1 2 h 1 in the black oat treatment and was significantly lower than that of the weed control plots at 0–5 cm depth. At 5–10 and 10– 15 cm soil depths, there were no significant differences between the weed control plots and the black oat plots. In plots where black oat was mixed with crimson clover, L-asparaginase activity increased considerably compared with that of black oat alone. Thus, in conventional tillage, crimson clover enhanced L-asparaginase activity. The observed increase in the L-asparaginase activity in the mixed plot originated from decomposition of crimson clover foliage and root biomass that enhanced the enzyme’s activity. In no-till plots, the enzyme activity was at all depths significantly (P 0.05) higher in the crimson clover plots than in the black oat or the black oat-crimson clover mixed plots (Fig. 2). The enzyme activity reached 11.7 mg NH4+–N kg 1 2 h 1 at 0– 5 cm depth and 8.73 mg NH4+–N kg 1 2 h 1 at 5–10 cm and 10– 15 cm depths in the crimson clover plots. Black oat in monoculture depressed activity of L-asparaginase activity as compared with the
3.2.2. Effects on L-glutaminase activity All the factors and their interactions significantly affected Lglutaminase activity in soil. In the tilled plots, L-glutaminase activity in the plots preceded by crimson clover significantly increased with depths from 42.2 mg NH4+–N kg 1 2 h 1 at 0–5 cm depth to 49.3 and 54.4 mg NH4+–N kg 1 2 h 1 at depths 5–10 cm and 10–15 cm, respectively (Fig. 3). At soil depths of 0–5 and 10– 15 cm, no significant differences (P 0.05) were found between activity of the enzyme in the black oat and black oat–crimson clover mixed tilled plots. Activity of the enzyme in the weed control plots was low at 0–5 cm depth (23.4 mg NH4+– N kg 1 2 h 1) and decreased with increasing soil depth to 19.3 and 15.4 mg NH4+–N kg 1 2 h 1 at 5–10 and 10–15 cm depth, respectively. Contrary to what was observed in the tilled plots, L-glutaminase activity under no-tilled plots preceded by crimson clover decreased from 67.9 mg NH4+–N kg 1 2 h 1 at depth 0–5 cm to 60.9 and 56.6 mg NH4+–N kg 1 2 h 1 at 5–10 cm and 10–15 cm depths, respectively (Fig. 3). However, at all depths the enzyme activity was significantly (P 0.05) greater in plots preceded by crimson clover than in those preceded by black oat or its mixture with crimson clover. Effects of cropping systems on four amidohydrolases (amidase, L-asparaginase, L-aspartase, and L-glutaminase) were reported in soils in Iowa and suggested that multicropping systems (meadowoat based systems) increased activities of the amidohydrolases compared with monocropping systems (Dodor and Tabatabai, 2003). Results of those studies suggested that those enzymes could be used to predict N mineralization and soil health. Our work forcefully showed that crimson clover stimulated L-glutaminase activity more than black oat. 3.2.3. Effects on urease activity Urease activity was significantly (P 0.05) affected only by the two-way interactions: cover crop soil sampling depth and tillage system soil sampling depth. In the tilled plots, no significant differences (P 0.05) were found among the treatments at all
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Fig. 4. Effect of cover crops on urease activity at different depths in conventional tillage and no-till systems.
Fig. 6. Relationships between urease activity and soil organic C (A), organic N (B) and between L-glutaminase activity and soil organic N (C). Opened circles are not included in the regression.
observed among crimson clover, black oat, and weed no-till plots. Urease activity at 10–15 cm under the crimson clover or black oat plot was significantly higher than that under the mixed plots. Our results suggested that urease activity may not be a good predictor of soil health in conventionally managed and no-till plots. Despite high coefficients of variation reported in a Walla soil (coarse-silty, mixed, mesic Typic Haploxeroll), application of N fertilizers significantly decreased urease activity while addition of manure increased its activity (Dick et al., 1988). The authors concluded that because the N fertilizers used in the experiments contained NH4+ and that the reaction products of urease being NH4+, microbial induction of urease activity had been inhibited. Similar studies conducted under laboratory conditions showed that ammoniumbased fertilizers had no effects on soil urease activity (Bremner and Mulvaney, 1978). 3.3. Relationships with organic carbon and nitrogen Fig. 5. Relationships between arylamidase activity and soil organic C (A), organic N (B) and between L-asparaginase activity and soil organic N (C). Opened circles are not included in the regression.
depths (Fig. 4). High coefficients of variation were reported for alkaline phosphatase (34%) and urease (29%) activities in longterm residue managed plots at 20 cm depth (Dick et al., 1988). However, in the no-till plots at 0–5 cm, the enzyme activity was significantly higher in plots preceded by crimson clover than in any others. At 5–10 cm soil depth, no significant differences were
Some of the enzymes studied significantly correlated with soil organic C and/or soil organic N. Arylamidase activity (Fig. 5A and B) significantly correlated with soil organic C (r = 0.699**) and soil organic N (r = 0.764***). Similarly, other studies have reported positive correlations between arylamidase activity and soil organic C (Acosta-Martı´nez and Tabatabai, 2001) and between arylamidase activity and soil pH (Acosta-Martı´nez and Tabatabai, 2001; Ekenler and Tabatabai, 2004). Green et al. (2007) reported significant correlation between arylamidase activity and soil total N and mineralized N.
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All amidohydrolases significantly correlated with soil organic N but only urease significantly correlated with soil organic C (r = 0.481*, Fig. 6A). The organic N correlated with L-asparaginase activity (r = 0.766***, Fig. 5C), with urease activity (r = 0.503*, Fig. 6B), and with L-glutaminase activity (r = 0.601**, Fig. 6C). The negative correlation between L-asparaginase activity and soil organic N implies that activity of the enzyme goes down as soil N content increases (Fig. 5C). Studies of Dick et al. (1988) have also shown negative correlations between enzyme (amidase, alkaline phosphatase, and urease) activities and N input. 4. Conclusions Our research demonstrated that enzyme activities were very sensitive to presence of cover crops and with the exception of urease in tilled plots, could be used as potential soil quality indicator. Individually used, crimson clover and black oat can stimulate arylamidase activity in till and no-till soils. L-Asparaginase activity was greater in till and no-till soils when tomato was preceded by crimson clover while, black oat completely depressed the enzyme’s activity in till and no-till plots. Similarly, activity of L-glutaminase was higher in soil when preceded by crimson clover than by black oat. Black oat is a grass that is usually used as a N scavenger in soil after summer legumes to prevent legume N from moving deep into the soil. Crimson clover on the other hand, is a legume that forms nodules in soil. These nodules are centers of intense N fixation activities. The NH4+ fixed is converted into asparagine, glutamine, glutamic acid, or other Nrich compounds that increase activities of responsible enzymes in soil and could explain the high activities of L-asparaginase and Lglutaminase in soil preceded by crimson clover. Although with the high dry-matter yields produced, the association crimson cloverblack oat seemed not to stimulate activities of the enzymes as much as when the cover crops are planted in monoculture. Our research suggested that management practices that incorporate cover crops into rotations, improve soil enzyme activities as compared with those systems which do not (weed fallows). Because the enzymes studied participate in the N cycling, their activities significantly correlated with soil organic N and C. References Acosta-Martı´nez, V., Tabatabai, M.A., 2000a. Enzyme activities of a limed agricultural soil. Biol. Fertil. Soils 31, 85–91.
Acosta-Martı´nez, V., Tabatabai, M.A., 2000b. Arylamidase activity of soils. Soil Sci. Soc. Am. J. 64, 215–221. Acosta-Martı´nez, V., Tabatabai, M.A., 2001. Tillage and residue management effects on arylamidase activity in soils. Biol. Fertil. Soils 34, 21–24. Benitez, E., Melgar, R., Sainz, H., Gomez, M., Nogales, R., 2000. Enzyme activities in the rhizosphere of pepper (Capsicum annuum L.) grown with olive cake mulches. Soil Biol. Biochem. 32, 1829–1835. Bremner, J.M., Keeney, D.R., 1966. Determination and isotope-ratio analysis of different forms of nitrogen in soils: 3. Exchangeable ammonium nitrate, and nitrite by extraction-distillation methods. Soil Sci. Soc. Am. Proc. 30, 577–582. Bremner, J.M., Mulvaney, R.L., 1978. Urease activity in soils. In: Burns, R.G. (Ed.), Soil Enzymes. Academic Press, New York, London, pp. 149–196. Chapman, H.D., 1965. Cation-exchange capacity. In: Black, C.A. (Ed.), Methods of Soil Analysis – Chemical and Microbiological Properties, Agronomy 9, 891– 901. Deng, S.P., Tabatabai, M.A., 1996a. Effect of tillage and residue management on enzyme activities in soils: I. Amidohydrolases. Biol. Fertil. Soils 22, 202–207. Deng, S.P., Tabatabai, M.A., 1996b. Effect of tillage and residue management on enzyme activities in soils: II. Glycosidases. Biol. Fertil. Soils 22, 208–213. Deng, S.P., Tabatabai, M.A., 1997. Effect of tillage and residue management on enzyme activities in soils: III. Phosphatases and arylsulfatase. Biol. Fertil. Soils 24, 141–146. Dick, R.P., 1994. Soil enzyme activities as indicators of soil quality. In: Doran, J.W., Coleman, D.C., Bezdicek, D.F., Stewart, B.A. (Eds.), Defining Soil for a Sustainable Environment. SSSA Spec. Pub. No. 35. SSSA, Madison, WI, pp. 3–21. Dick, R.P., Rasmussen, P.E., Kerle, E.A., 1988. Influence of long-term residue management on soil enzyme activities in relation to soil chemical properties of a wheatfallow system. Biol. Fertil. Soils 6, 159–164. Dodor, D.E., Tabatabai, M.A., 2003. Amidohydrolases in soils as affected by cropping systems. Appl. Soil Ecol. 24, 73–90. Ekenler, M., Tabatabai, M.A., 2004. Arylamidase and amidohydrolases in soils as affected by liming and tillage systems. Soil Till. Res. 77, 157–168. Frankenberger Jr., W.T., Dick, W.A., 1983. Relationships between enzyme activities and microbial growth and activity indices in soil. Soil Sci. Soc. Am. J. 47, 945– 951. Green, V.S., Stott, D.E., Cruz, J.C., Curi, N., 2007. Tillage impacts on soil biological activity and aggreration in a Brazilian Cerrado Oxisol. Soil Till. Res. 92, 114– 121. Hiwada, K., Yamaguchi, C., Inaoka, Y., Kokubu, T., 1977. Neutral arylamidase in urine of healthy and nephrotic children. Clin. Chim. Acta 75, 31–39. Jarrel-Ash Division, 1982. Jarrel-Ash ICAP-9000 Plasma Spectrotometer Operator’s Manuel Jarrel-Ash Division, Fisher Scientific Co, Franklin, Massachusetts. Mehlich, A., 1978. New extractant for soil test evaluation of phosphorus, potassium, magnesium, calcium, sodium, manganese, and zinc. Commun. Soil Sci. Plant Anal. 9 (6), 477–492. Nannipieri, P., Kandeler, E., Ruggiero, P., 2002. Enzyme activities and microbiological and biochemical processes in soil. In: Burns, R.G., Dick, R.P. (Eds.), Enzymes in the Environment. Activity, Ecology and Applications. Dekker, New York, pp. 1–33. Olsen, S.R., Sommers, L.E., 1982. Phosphorus. In: Methods of Soil Analysis, Part 2. Soil Sci. Soc. Am., Madison, WI, pp. 403–430. SAS Institute, 2008. SAS user’s guide, North Carolina. Tabatabai, M.A., 1994. Soil enzymes. In: Weaver, R.W., Angel, J.S., Bottomley, P.S. (Eds.), Methods of Soil Analysis, Part 2, Book Series 5. Soil Sci. Soc. Am., Madison, WI, pp. 775–833. Tate III, R.L., 1987. Soil enzymes and organic matter transformations. In: Soil Organic Matter; Biological and Ecological Effects, Wiley, pp. 69–94.
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Cover crop and tillage effects on soil enzyme activities following tomato Said A. Hamido, K. Kpomblekou-A * Department of Agricultural and Environmental Sciences, Tuskegee University, 213 Milbank Hall, Tuskegee, AL 36088, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 May 2009 Received in revised form 4 September 2009 Accepted 12 September 2009
Increasing numbers of vegetable growers are adopting conservation tillage practices and including cover crops into crop rotations. The practice helps to increase or maintain an adequate level of soil organic matter and improves vegetable yields. The effects of the practices, however, on enzyme activities in southeastern soils of the United States have not been well documented. Thus, the objectives of the study were to investigate the effects of cover crops and two tillage systems on soil enzyme activity profiles following tomato and to establish relationships between enzyme activities and soil organic carbon (C) and nitrogen (N). The cover crops planted late in fall 2005 included black oat (Avena strigosa), crimson clover (Trifolium incarnatum L.), or crimson clover–black oat mixed. A weed control (no cover crop) was also included. Early in spring 2006, the plots were disk plowed and incorporated into soil (conventional tillage) or mowed and left on the soil surface (no-till). Broiler litter as source of N fertilizer was applied at a rate of 4.6 Mg ha 1, triple super phosphate at 79.0 kg P ha 1, and potassium chloride at 100 kg K ha 1 were also applied according to soil testing recommendations. Tomato seedlings were transplanted and grown for 60 days on a Marvyn sandy loam soil (fine-loamy, kaolinitic, thermic Typic Kanhapludults). Ninety-six core soil samples were collected at incremental depths (0–5, 5–10, and 10–15 cm) and passed through a 2-mm sieve and kept moist to study arylamidase (EC 3.4.11.2), L-asparaginase (EC 3.5.1.1), L-glutaminase (EC 3.5.1.2), and urease (EC 3.5.1.5) activities. Tillage systems affected only L-glutaminase activity in soil while cover crops affected activities of all the enzymes studied with the exception of urease. The research clearly demonstrated that in till and no-till systems, L-asparaginase activity is greater (P 0.05) in plots preceded by crimson clover than in those preceded by black oat or their mixture. Activity of the enzyme decreased from 11.7 mg NH4+–N kg 1 2 h 1 at 0–5 cm depth to 8.73 mg NH4+–N kg 1 2 h 1 at 5–10 cm and 10–15 cm depths in the no-till crimson clover plots. Arylamidase activity significantly correlated with soil organic C (r = 0.699**) and soil organic N (r = 0.764***). Amidohydrolases activities significantly correlated with soil organic N but only urease significantly correlated with soil organic C (r = 0.481*). These results indicated that incorporation of cover crops into rotations may increase enzyme activities in soils. ß 2009 Published by Elsevier B.V.
Keywords: Enzyme activities Conventional tillage No-till Cover crops Broiler litter
1. Introduction Soils are inhabited by a vast array of microbes responsible for breakdown of organic matter and solubilization of nutrients. These microbes are major source of soil enzymes and their dynamics in soils seemed to be related to management practices. Enzymes play an important role in the cycling of nutrients in nature and because soil enzyme activity is sensitive to agricultural practices, it can be used as an index of soil microbial activity and fertility (Benitez et al., 2000). Thus, they are involved in soil mineralization processes (Tate, 1987) and have been related to other soil biological properties (Frankenberger and Dick, 1983). Enzyme activity profiles reflect an essential part of soil functional diversity,
* Corresponding author. Tel.: +1 334 724 4521; fax: +1 334 724 4529. E-mail address: [email protected] (K. Kpomblekou-A). 0167-1987/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.still.2009.09.007
which is controlled by genetic diversity of soil microorganisms, plants and soil animals in close relation to environmental effects and ecological interactions (Nannipieri et al., 2002). Enzymes catalyze a complex web of chemical reactions necessary for decomposition of organic residues, cycling of nutrients, formation of organic matter, and soil structure (Dick, 1994). Amino acid arylamidase and several amidohydrolases have been identified in soils (Acosta-Martı´nez and Tabatabai, 2001). The most important of the enzymes that play significant role in N mineralization are arylamidase, L-asparaginase, L-glutaminase, amidase, and urease. Because enzymes provide a useful tool for long-term monitoring of changes in soil health and quality, they have been proposed as soil fertility indicators (Frankenberger and Dick, 1983). Arylamidase (EC 3.4.11.2) is an enzyme that catalyzes the hydrolysis of an N-terminal amino acid from peptides amides using L-leucine as the amino acid moiety and its activity was detected in soils (Acosta-Martı´nez and Tabatabai, 2000a,b). This
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enzyme catalyzes one of the most important reactions in N mineralization. It releases amino acids from soil organic matter that are used as substrates for amidohydrolases. Previous studies demonstrated that the activity of arylamidase is significantly affected by crop residue managements (Deng and Tabatabai, 1996a,b, 1997) and tillage systems (Acosta-Martı´nez and Tabatabai, 2001). Acosta-Martı´nez and Tabatabai (2001) compared enzyme activities in soil samples collected from no-till/double mulch and tillage with residue management treatment plots. The authors reported the greatest enzyme activity in the no-till/double mulch plot. Amidohydrolases are widely distributed in nature and have been detected in plants, animals, and microorganisms (Tabatabai, 1994). Soil samples (0–15 cm) obtained from Northeast Research Center in Nashua (Iowa) for a period of seven years (1987–1994) demonstrated that liming increased activities of 13 enzymes (Acosta-Martı´nez and Tabatabai, 2000a). Previous studies on the effect of tillage systems and residue management showed that activities of L-asparaginase, L-glutaminase, and urease were generally greater under no-till systems than under conventional tillage systems (Deng and Tabatabai, 1996a,b, 1997). Activities of phosphatases (acid phosphatase, alkaline phosphatase, phosphodiesterase, and inorganic pyrophosphatase) and arylsulfatase were investigated in three tillage systems (no-till, chisel plow, and moldboard plow) in combination with corn residue placements. Activities of the enzymes in no-till/double mulch were significantly greater than those of other treatments studied (Deng and Tabatabai, 1996a). The activities decreased with increasing soil depths and the decreases were associated with a decrease in organic carbon (C) content. Because soil enzyme activities respond to external organic matter inputs and tillage systems, it is important to investigate activities of enzymes directly responsible for nitrogen (N) transformations in soils when legumes and grasses are added to soil under different tillage systems. Thus, understanding profile distribution of these enzymes in soil will be a valuable tool in evaluating the effect of cover crops on N dynamics in soils. The objectives of the present study were to: (i) evaluate under a conventional (disk plow) and a no-till system, soil enzymes activities in tomato plots preceded by crimson clover, black oat, or their mixture and (ii) establish relationships between enzyme activities and soil organic N and/or organic C. 2. Materials and methods The experiment was initiated in fall 2005 at the George Washington Carver Agricultural Experiment Station at Tuskegee University, Tuskegee, AL, on a Marvyn sandy loam (fine-loamy, kaolinitic, thermic Typic Kanhapludults) soil. Prior to establishment of the experiment, the site was in fallow (mainly grasses) for several years. Composite soil samples were collected from 0 to 15 cm depth for soil characterization and fertilizer recommendations. 2.1. Soil analysis and trial description Soil pH (6.1) was determined by a glass electrode (soil:water 1:2.5) and soil cation exchange capacity (<4.6 cmolc kg 1) was determined by using a neutral 1 M NH4OAc solution as described by Chapman (1965). Extractable P (10.0 mg kg 1) was determined as outlined by Olsen and Sommers (1982), while extractable K (78.0 mg kg 1), Mg (73.0 mg kg 1), and Ca (350 mg kg 1) were measured as described by Mehlich (1978). Extractable metals in the solution were determined by using Inductively Coupled Argon Plasma (ICAP) in a Thermo Jarrell Ash, Model 9000 (Jarrel-Ash Division, 1982). Ammonium–N and nitrate (NO3 + NO2 )–N contents of the soil were extracted with 2 M KCl solution and
determined by steam distillation (Bremner and Keeney, 1966) and averaged 2.14 and 1.65 mg kg 1 soil, respectively. The cover crops tested were crimson clover (Trifolium incarnatum L.), black oat (Avena strigosa), crimson clover–black oat mixed, and a weed–control plot without cover crop. The cover crops were planted late in fall 2005, cut and incorporated into soil by disk plow (conventional tillage) or left on the soil surface as a mulch (no-till) late in the following spring. Broiler litter at a rate of 4.6 Mg ha 1, triple super phosphate at 79.0 kg P ha 1, and potassium chloride at 100 kg K ha 1 were applied to all treatments. Tomato (Lycopersicon esculentum Mill.) seedlings were transplanted and grown for 60 days. Following tomato harvest, ninety-six core soil samples (2.5 cm, ID) were collected at incremental depths (0–5, 5–10, and 10– 15 cm) using an auger to investigate arylamidase and amidohydrolases activities in each experimental unit (3 5 m). The experimental design was a split plot with four replications. The four cover crops were randomly assigned to the horizontal strips and the two tillage systems (conventional tillage or no-till) were randomly assigned to the vertical strips. 2.2. Enzyme activities The soil samples were passed through a 2-mm sieve and kept moist in a refrigerator at 4 8C until used. Arylamidase activity was assayed by incubating 1 g moist soil sample with 3 mL of 0.1 M THAM buffer (pH 8.0) and 1 mL 8.0 mM solution of L-leucine bnaphthylamide hydrochloride (Acosta-Martı´nez and Tabatabai, 2000b). The product of the reaction b-naphthylamine was measured at 540 nm (Hiwada et al., 1977) using a Beckman Coulter DU 640 spectrophotometer (Beckman Instruments, Inc. Fullerton, CA). Controls were included as described for the assay with the exception that the substrate L-leucine b-naphthylamide hydrochloride was added after incubation. Amidohydrolases (L-asparaginase, L-glutaminase, and urease) activities were assayed by incubating 5 g moist soil sample with 1 mL 0.5 M solution of L-asparagine, L-glutamine, or 0.2 M solution of urea and 9 mL of THAM [Tris(hydroxymethyl)aminomethane] buffer in the presence of 0.2 mL of toluene (Tabatabai, 1994). The ammonia formed following hydrolysis of L-asparaginase, Lglutaminase, or urease was measured by steam distillation using 20 mL aliquot of the soil suspension and 0.2 g magnesium oxide (MgO) as described by Bremner and Keeney (1966). Appropriate controls were included in all the assays to account for NH4+–N not derived from L-asparagine, L-glutamine, or urea through their respective enzyme activity (Tabatabai, 1994). All results reported are averages of duplicate assays expressed on moisture-free basis. Moisture was determined from loss in weight following drying at 105 8C for 48 h. 2.3. Cover crop biomass Before cutting the cover crops, four areas of 1 m2 were randomly marked on each experimental unit. The plant material within each marked area was cut above-ground and weighed to estimate the mean cover crop biomass of each treatment. A subsample was taken in a brown bag, weighed, transported to the laboratory, and placed in an oven at 65 8C for 72 h to determine the dry-matter yield while the remaining above-ground plant samples were returned to each corresponding plot. 2.4. Statistical analysis Because measures obtained at the three depths are not independent, data were analyzed using MIXED procedure of SAS (SAS ver. 9.1, Institute, Cary, NC, USA) with a repeated measure
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statement that assumed unstructured (co) variances. Denominator degrees of freedom were calculated using the Kenward–Roger option. Means were compared with Fisher’s protected LSD. The SLICE option and individual degree of freedom contrasts were used to explain significant interactions. The level of significance was set at 0.05. 3. Results and discussion In general, cover crops and soil sampling depths significantly (P 0.05) affected arylamidase, L-asparaginase, and L-glutaminase activities in soil. Urease activity was not significantly affected by the presence of cover crops, tillage systems and soil sampling depths. The two-way interactions: cover crop tillage system and tillage system soil sampling depth were the only combination of factors that significantly affected urease activity in soil. The interaction tillage system soil sampling depth was also significant for L-glutaminase activity while the three-way interaction significantly affected only L-asparaginase and L-glutaminase activities. The above-ground cover crops biomass dry-matter yields incorporated in the soils are shown in Table 1. There were no significant differences (P 0.05) among the cover crops tested in term of dry-matter yields produced but the enzyme activities induced by the cover crops varied significantly. 3.1. Effects of cover crops and tillage systems on arylamidase activity Statistical analyses suggested that cover crops and soil sampling depths affected significantly (P 0.05) arylamidase activity (Fig. 1). The average of arylamidase activity in the notill plots was 7.94 mg b-naphthylamine kg 1 h 1 and 7.34 mg bnaphthylamine kg 1 h 1 on the tilled plots. Across all cover crops, the activities were 7.36 and 8.67 mg b-naphthylamine kg 1 h 1 at 0–5 cm depth, 7.81 and 8.38 mg b-naphthylamine kg 1 h 1 at 5– 10 cm depth, and 6.84 and 6.77 mg b-naphthylamine kg 1 h 1 at 10–15 cm depth in the tilled and no-tilled treatments, respectively. Tillage practices by themselves did not significantly affect arylamidase activity in the soil. Fig. 1 showed that at 0–5 cm soil depth, arylamidase activity in the tilled plots was significantly (P 0.05) higher (10.9 mg bnaphthylamine kg 1 h 1) in the black oat plot than in the weed plot (3.13 mg b-naphthylamine kg 1 h 1) or the crimson clover– black oat mixed (7.43 mg b-naphthylamine kg 1 h 1). At 5–10 cm depth, there was no significant difference (P 0.05) between arylamidase activity observed under black oat, crimson clover, or their mixed treatment plots in the conventional tillage plots. However, the three treatments showed higher arylamidase activity than the weed control tilled plot. In addition, at 10–15 cm depth, there was no significant difference among the black oat, the crimson clover, and the mixed plots; the activity of arylamidase was however, greater in those plots than in the control tilled weed plots where the activity was only 3.38 mg b-naphthylamine kg 1 h 1. The activity of arylamidase increased in the tilled plot under crimson clover from 0–5 to 5–10 cm depth but decreased at 10–15 cm. The activity decreased with depths in plots preceded by black oat. Under the crimson clover–black oat mixed tilled plot, the enzyme activity remained constant (7.5 mg b-naphthylamine
Fig. 1. Effect of cover crops on arylamidase activity at different depths in conventional tillage and no-till systems.
kg 1 h 1), the weed control plots also showed constant activities (3.3 mg b-naphthylamine kg 1 h 1) with depths. This suggests that the cover crops increased activity of arylamidase in soil and that microbial respiration was likely higher in the upper plow layer of the soil where black oat was incorporated. Generally, enzyme activities tend to decrease with depths and differentiation among treatments are found often only in the 0–5 cm depth (Ekenler and Tabatabai, 2004; Green et al., 2007). In a Brazilian Cerrado Oxisol, arylamidase and acid phosphatase activities were 18–186% greater under no-till than under disk plow in 0–5 cm depth (Green et al., 2007). However, in a no-till soil, Ekenler and Tabatabai (2004) reported an increase in arylamidase activity from 16.1 at 0–5 cm depth to 19.8 mg b-naphthylamine kg 1 h 1 at 0–15 cm depth. Our results clearly suggested that arylamidase activity is influenced by the presence of crimson clover or black oat and therefore, could be used to monitor soil health and quality under the conditions of this experiment as suggested by previous studies (Dick, 1994). In the no-till plots, arylamidase activity was consistently higher under black oat at 5–10 and 10–15 cm soil depths than under crimson clover or mixed with crimson clover (Fig. 1). The activities under the black oat treatments were 12.6, 12.6, and 10.5 mg bnaphthylamine kg 1 h 1 at 0–5, 5–10, and 10–15 cm, respectively and were significantly (P 0.05) greater than those under crimson clover and the mixture at 10–15 cm depth. Although the activities of arylamidase under black oat remained high at all soil depths, those under crimson clover decreased steadily with depths to 7.18 mg b-naphthylamine kg 1 h 1 at 10–15. Black oat mulch on top of the soil induced arylamidase activity in the top 0–10 cm but the activity was reduced at 10–15 cm depth although not significantly. A slow decomposition of this mulch increased microbial activity contributing to a higher amount of endoenzymes in the viable microbial populations in the topsoil. It appeared like crimson clover significantly depressed activity of arylamidase in the mixture at all depths. Green et al. (2007) reported greater activity of arylamidase under no-till and disk harrow than under
Table 1 Cover crop biomass yields. Cover crop
Fresh yield (Mg ha
Weed Crimson clover Black oat Crimson clover–black oat mixture
2.78 3.35 3.59 5.20
1
)
b ab ab a
Values in the same column followed by the same letter are not significantly different at P 0.05.
Moisture content (%)
Dry-matter yield (Mg ha
76.9 62.9 57.4 59.2
0.64 1.24 1.53 2.12
b ab a a
1
)
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Fig. 2. Effect of cover crops on L-asparaginase activity at different depths in conventional tillage and no-till systems.
Fig. 3. Effect of cover crops on L-glutaminase activity at different depths in conventional tillage and no-till systems.
disk plow. Studies by Ekenler and Tabatabai (2004) also demonstrated greater arylamidase activity in no-till plots than in ridgetill and chisel plow plots at Northwest Research Center site at Sutherland (Iowa, U.S.A.). Our data support previous works and demonstrated that in no-till systems, enzyme activities tend to be stratified in soil profiles and implied that in some cases, to detect enzyme activities, samples must be taken incrementally to avoid a dilution effect.
weed control plots that showed higher activity of the enzyme. In no-till plots, the enzyme activity did not improve even in the presence of crimson clover in the mixed plots. It was evident that black oat significantly depressed L-asparaginase activity in no-till soils. One might suggest that black oat, because of its allelopathic properties, released substances that inhibited activity of the enzyme.
3.2. Effects of cover crops and tillage systems on amidohydrolases activity Tillage practices have profound influences on amidohydrolases activity in soils. Deng and Tabatabai (1996a) showed that activities of amidohydrolases were generally greater in mulch-treated plots than in non-treated plots and were related to soil organic C. In the present studies, activities of the enzymes varied with cover crops and soil depths. 3.2.1. Effects on L-asparaginase activity In general, tillage practices did not have a significant effect on Lasparaginase activity; however, its two-way interaction with cover crops or with soil sampling depths, and three-way interaction were significant. L-Asparaginase activity in plots preceded by crimson clover was higher than that of plots preceded by black oat at all depths and tillage systems (Fig. 2). Under conventional tillage, the activity of L-asparaginase was as low as 0.75 mg NH4+–N kg 1 2 h 1 in the black oat treatment and was significantly lower than that of the weed control plots at 0–5 cm depth. At 5–10 and 10– 15 cm soil depths, there were no significant differences between the weed control plots and the black oat plots. In plots where black oat was mixed with crimson clover, L-asparaginase activity increased considerably compared with that of black oat alone. Thus, in conventional tillage, crimson clover enhanced L-asparaginase activity. The observed increase in the L-asparaginase activity in the mixed plot originated from decomposition of crimson clover foliage and root biomass that enhanced the enzyme’s activity. In no-till plots, the enzyme activity was at all depths significantly (P 0.05) higher in the crimson clover plots than in the black oat or the black oat-crimson clover mixed plots (Fig. 2). The enzyme activity reached 11.7 mg NH4+–N kg 1 2 h 1 at 0– 5 cm depth and 8.73 mg NH4+–N kg 1 2 h 1 at 5–10 cm and 10– 15 cm depths in the crimson clover plots. Black oat in monoculture depressed activity of L-asparaginase activity as compared with the
3.2.2. Effects on L-glutaminase activity All the factors and their interactions significantly affected Lglutaminase activity in soil. In the tilled plots, L-glutaminase activity in the plots preceded by crimson clover significantly increased with depths from 42.2 mg NH4+–N kg 1 2 h 1 at 0–5 cm depth to 49.3 and 54.4 mg NH4+–N kg 1 2 h 1 at depths 5–10 cm and 10–15 cm, respectively (Fig. 3). At soil depths of 0–5 and 10– 15 cm, no significant differences (P 0.05) were found between activity of the enzyme in the black oat and black oat–crimson clover mixed tilled plots. Activity of the enzyme in the weed control plots was low at 0–5 cm depth (23.4 mg NH4+– N kg 1 2 h 1) and decreased with increasing soil depth to 19.3 and 15.4 mg NH4+–N kg 1 2 h 1 at 5–10 and 10–15 cm depth, respectively. Contrary to what was observed in the tilled plots, L-glutaminase activity under no-tilled plots preceded by crimson clover decreased from 67.9 mg NH4+–N kg 1 2 h 1 at depth 0–5 cm to 60.9 and 56.6 mg NH4+–N kg 1 2 h 1 at 5–10 cm and 10–15 cm depths, respectively (Fig. 3). However, at all depths the enzyme activity was significantly (P 0.05) greater in plots preceded by crimson clover than in those preceded by black oat or its mixture with crimson clover. Effects of cropping systems on four amidohydrolases (amidase, L-asparaginase, L-aspartase, and L-glutaminase) were reported in soils in Iowa and suggested that multicropping systems (meadowoat based systems) increased activities of the amidohydrolases compared with monocropping systems (Dodor and Tabatabai, 2003). Results of those studies suggested that those enzymes could be used to predict N mineralization and soil health. Our work forcefully showed that crimson clover stimulated L-glutaminase activity more than black oat. 3.2.3. Effects on urease activity Urease activity was significantly (P 0.05) affected only by the two-way interactions: cover crop soil sampling depth and tillage system soil sampling depth. In the tilled plots, no significant differences (P 0.05) were found among the treatments at all
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Fig. 4. Effect of cover crops on urease activity at different depths in conventional tillage and no-till systems.
Fig. 6. Relationships between urease activity and soil organic C (A), organic N (B) and between L-glutaminase activity and soil organic N (C). Opened circles are not included in the regression.
observed among crimson clover, black oat, and weed no-till plots. Urease activity at 10–15 cm under the crimson clover or black oat plot was significantly higher than that under the mixed plots. Our results suggested that urease activity may not be a good predictor of soil health in conventionally managed and no-till plots. Despite high coefficients of variation reported in a Walla soil (coarse-silty, mixed, mesic Typic Haploxeroll), application of N fertilizers significantly decreased urease activity while addition of manure increased its activity (Dick et al., 1988). The authors concluded that because the N fertilizers used in the experiments contained NH4+ and that the reaction products of urease being NH4+, microbial induction of urease activity had been inhibited. Similar studies conducted under laboratory conditions showed that ammoniumbased fertilizers had no effects on soil urease activity (Bremner and Mulvaney, 1978). 3.3. Relationships with organic carbon and nitrogen Fig. 5. Relationships between arylamidase activity and soil organic C (A), organic N (B) and between L-asparaginase activity and soil organic N (C). Opened circles are not included in the regression.
depths (Fig. 4). High coefficients of variation were reported for alkaline phosphatase (34%) and urease (29%) activities in longterm residue managed plots at 20 cm depth (Dick et al., 1988). However, in the no-till plots at 0–5 cm, the enzyme activity was significantly higher in plots preceded by crimson clover than in any others. At 5–10 cm soil depth, no significant differences were
Some of the enzymes studied significantly correlated with soil organic C and/or soil organic N. Arylamidase activity (Fig. 5A and B) significantly correlated with soil organic C (r = 0.699**) and soil organic N (r = 0.764***). Similarly, other studies have reported positive correlations between arylamidase activity and soil organic C (Acosta-Martı´nez and Tabatabai, 2001) and between arylamidase activity and soil pH (Acosta-Martı´nez and Tabatabai, 2001; Ekenler and Tabatabai, 2004). Green et al. (2007) reported significant correlation between arylamidase activity and soil total N and mineralized N.
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All amidohydrolases significantly correlated with soil organic N but only urease significantly correlated with soil organic C (r = 0.481*, Fig. 6A). The organic N correlated with L-asparaginase activity (r = 0.766***, Fig. 5C), with urease activity (r = 0.503*, Fig. 6B), and with L-glutaminase activity (r = 0.601**, Fig. 6C). The negative correlation between L-asparaginase activity and soil organic N implies that activity of the enzyme goes down as soil N content increases (Fig. 5C). Studies of Dick et al. (1988) have also shown negative correlations between enzyme (amidase, alkaline phosphatase, and urease) activities and N input. 4. Conclusions Our research demonstrated that enzyme activities were very sensitive to presence of cover crops and with the exception of urease in tilled plots, could be used as potential soil quality indicator. Individually used, crimson clover and black oat can stimulate arylamidase activity in till and no-till soils. L-Asparaginase activity was greater in till and no-till soils when tomato was preceded by crimson clover while, black oat completely depressed the enzyme’s activity in till and no-till plots. Similarly, activity of L-glutaminase was higher in soil when preceded by crimson clover than by black oat. Black oat is a grass that is usually used as a N scavenger in soil after summer legumes to prevent legume N from moving deep into the soil. Crimson clover on the other hand, is a legume that forms nodules in soil. These nodules are centers of intense N fixation activities. The NH4+ fixed is converted into asparagine, glutamine, glutamic acid, or other Nrich compounds that increase activities of responsible enzymes in soil and could explain the high activities of L-asparaginase and Lglutaminase in soil preceded by crimson clover. Although with the high dry-matter yields produced, the association crimson cloverblack oat seemed not to stimulate activities of the enzymes as much as when the cover crops are planted in monoculture. Our research suggested that management practices that incorporate cover crops into rotations, improve soil enzyme activities as compared with those systems which do not (weed fallows). Because the enzymes studied participate in the N cycling, their activities significantly correlated with soil organic N and C. References Acosta-Martı´nez, V., Tabatabai, M.A., 2000a. Enzyme activities of a limed agricultural soil. Biol. Fertil. Soils 31, 85–91.
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