Quality of soil organic matter and C storage as

NRC Publications Archive (NPArC) Archives des publications du CNRC (NPArC) Quality of soil organic matter and C storage as influenced by cropping systems in northwestern Alberta, Canada Arshad, M. A.; Soon, Y. K.; Ripmeester, J. A

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Nutr Cycl Agroecosyst (2011) 89:71–79 DOI 10.1007/s10705-010-9377-1

ORIGINAL ARTICLE

Quality of soil organic matter and C storage as influenced by cropping systems in northwestern Alberta, Canada M. A. Arshad • Y. K. Soon • J. A. Ripmeester

Received: 22 February 2010 / Accepted: 22 May 2010 / Published online: 4 June 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Crop rotations and reduction in tillage are commonly recommended for sustained crop production and enhancing soil quality. Our objective was to evaluate the long-term effects of cropping systems (1968–1992) on soil structure, carbon storage and the quality of soil organic matter. The study was conducted on a silt clay loam soil (Typic Cryoboralf) near Beaverlodge, Alberta, The cropping systems were: (a) continuous barley (Hordeum vulgare L.) (CB); (b) continuous bromegrass (Bromus inermiss Leyess.) (CG); (c) continuous forage legume (Medicago sativa L. until 1977, and Trifolium pratense L. since 1978) (CL); and (d) 3 years of bromegrasslegume forage alternating with 3 years of barley (RF). Our data showed that the CG and CL treatments had more stable aggregates with greater mean weight diameter (MWD) than soil under continuous barley. Organic C, total N and the light fraction in soil under CG and CL were higher than those of the other two treatments. Soil under CG had the highest and CB the lowest amounts of acid-hydrolyzable monosacchrides (comprising glucose, arabinose, xylose, mannose and galactose). Higher galactose ? mannose M. A. Arshad (&) Y. K. Soon Research Branch, Agriculture and Agri-Food Canada, P. O. Box 29, Beaverlodge, AB, Canada T0H 0C0 e-mail: [email protected] J. A. Ripmeester Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON, Canada K1A 0R6

concentration in soil under CG indicated a higher soil microbiological activity. Microbial biomass C and N followed the trend among treatments in whole and light fraction organic matter, and total extracted sugars. Soil organic matter 13C-NMR spectroscopy showed that: (i) soil under CB contained the highest amounts of aromatic and the lowest content of aliphatic-C, (ii) soil under CL had the lowest phenolic-C and the least aromaticity, and (iii) soil under CG and RF had the highest amounts of aliphatic-C which includes labile substances such as amino acids and carbohydrates, indicating an improvement in the quality of organic matter. It is concluded that perennial forage crops can improve soil structure and soil organic matter quality and quantity as compared with cereal monoculture. Keywords Aggregation Carbohydrates C-NMR spectroscopy Carbon storage Light fraction organic matter Microbial biomass 13

Introduction Alterations in the quantity and composition of soil organic matter (SOM) under different cropping systems can have a significant impact on the quality of a soil. Depending on crop species and sequences used, crop rotations generally result in higher soil organic matter contents than cereal monoculture (Mannan 1962; Roder et al. 1988). Effects of

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cropping systems on soil organic matter have been reported by several researchers (Campbell et al. 1990; Janzen 1987; Janzen et al. 1992, 1998). Organic matter was higher, and the C:N ratio wider, in rotation plots when sorghum was included in the cropping system (Mannan 1962). Excessive tillage and use of summer fallow in farming systems, practiced in many semi-arid regions of the world, reduce inputs of plant residues, increase decomposition rates, break down stable aggregates, and increase soil erosion (Elliott 1986; Karlen et al. 1994; Bremer et al. 1995). On the other hand, soil and crop management systems that include forages and legumes, cover crops, and no-till practices improve soil structure (aggregation) and soil organic matter (Arshad et al. 1990, 2004; Alvaro-Fuentes et al. 2009; Limon-Ortega et al. 2009). The influence of crop rotations on soil structure and agronomic parameters has been recently reviewed by Ball et al. (2005). On the Canadian prairies, numerous studies have been conducted to assess the long-term effects of crop rotations on crop yields and soil properties including changes in soil organic matter (Janzen et al. 1992, 1998. However, limited information is available on the impact of long-term crop rotations on structural properties and the quality of organic components of soils of northwestern Canada. The objectives of this study were: to investigate changes in soil structure and the quality of SOM, and determine differences, if any, in carbon storage under various long-term crop rotations in the cold semi-arid climate of northern Alberta.

Materials and methods Site characteristics The experimental site was located at the Agriculture and Agri-food Canada research farm in Beaverlodge, Alberta (approximately (55°130 N, 119°200 W). The soil at the study site was poorly drained, developed on lacustro-till parent material and is classified as a Dark Grey Luvisol in the Canadian classification (Canada Department of Agriculture 1978), an Albic Luvisol according to the FAO (FAO/UNESCO 1988) and a fine montmorillonitic, frigid, Mollic Cryoboralf in the USDA taxanomy (Soil Survey Staff 1994). The top 75-mm of soil contained 200 g sand kg-1, 270 g

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Nutr Cycl Agroecosyst (2011) 89:71–79

clay kg-1, and had a pH (1:2, soil: 0.01 M CaCl2) of 5.1, and a cation exchange capacity of 21 cmolc kg-1 (measured by summation of exchangeable cations, as reported by Soon and Arshad 1996). Annual temperature and precipitation average 2°C and 452 mm, respectively. Long-term 30-year (1971– 2000) means of the growing season (May–August) are: precipitation 247 mm, class A pan evaporation 831 mm, temperature 13.3°C and growing degree days ([5°C) 1,224°C degree-days. Experimental design The cropping history and details of treatments have been described previously (Soon and Arshad 1996; Soon 1998). Briefly, the treatments for this study were started in 1968 and consisted of: (a) continuous barley (Hordeum vulgare L.) (CB); (b) continuous bromegrass (Bromus inermiss Leyess) (CG); (c) continuous forage legume (Medicago sativa L. until 1977, and Trifolium pratense L. since 1978) (CL); and (d) 3 years of bromegrass-legume forage alternating with 3 years of barley (RF) with both phases, barley and forages, represented in any 1 year. The final year of barley phase of the rotation was underseeded with bromegrass-legume so that the forage phase was in production the following year. Each treatment was replicated four times in a randomized complete block design with plots measuring 58 m by 22 m. The rotation forage was terminated by moldboard ploughing in late summer or early autumn. Seedbed preparation for sowing barley the following spring was typically done with one or two passes with tandem discs followed by harrow and packer. For barley following barley (CB) or the barley phase of the forage/barley rotation (RF), tillage operations consisted of one pass with a heavy cultivator followed by tandem discs. Continuous grass or legume was reseeded whenever the stand had deteriorated significantly. Soil sampling and analyses Undisturbed soil cores, 75 mm in diameter and 75 mm long, were taken in 1992 (spring) from each of the four replicate plots to characterize the soil properties. For the RF rotation treatment, soil samples were taken from plots under barley phase. Soil for organic C and total N analysis was air dried and


ground to pass through 60-mesh sieve. Organic carbon was determined, using the modified Mebius method, in digestion tubes heated in aluminum blocks (Nelson and Sommers 1982). Total nitrogen was determined by automated colorimetry (indophenol blue method) following Kjeldahl digestion in 100-ml digestion tubes with K2SO4-Cu catalyst in heated aluminum block. A separate set of undisturbed (prior to seeding operations) soil cores were taken from each treatment in spring 1992 for bulk density. Soil bulk density was calculated from an oven dried weight of soil and volume of the soil core. Unfortunately, soil bulk density could not be determined in the year of initiation of this study in 1968 when soil samples were collected, air-dried, ground to pass 2 mm sieve and archived. Thus, all soil chemical analyses for the 1968 year were done on the archived samples. Aggregate size distribution was determined from 50 g subsamples of air-dried soil directly immersed in water for 10 min with a stroke length of 35 mm and a frequency of 16 cycles min-1. Dry weight (50°C) of soil retained on sieves with openings of 4, 2, 1, 0.5, 0.25, and 0.13 mm was used to determine the mean-weight diameter. The mean weight diameter (MWD) was calculated by summation of the mean diameter of each size fraction multiplied by the fractional weight of each size fraction (Kemper and Rosenau 1986). The fraction\0.13 was calculated as the difference between initial and the sum of other six size-fractions. The light-fraction of organic matter was determined by the method described by Strickland and Sollins (1987), as modified by Janzen et al. (1992). The procedure consisted of the dispersion of a soil sample (\2 mm, air-dried) in a NaI solution with a specific gravity of 1.7, and the separation of suspended material, i.e., the light fraction, floating on the liquid surface. The C and N concentrations of the light fraction materials were determined by standard wet digestion methods as described above. Soil monosaccharides were extracted using 1.5 M H2SO4 (85°C for 24 h) and quantified by HPLC according to Angers et al. (1988). Soil microbial C and N were extracted in 0.25 M K2SO4 from field moist soil samples by the fumigation-extraction methods outlined by Vance et al. (1987) and Brookes et al. (1985), respectively. Extracted C was determined by titration following dichromate digestion; extracted N underwent Kjeldahl digestion and was determined by automated colorimetry as ammonium-N.

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Samples from four replicates of each treatment were composited for 13C NMR spectroscopy. Solid state 13C NMR spectra were recorded at a frequency of 50 MHz on a Bruker CXP-180 NMR spectrometer equipped with 5 mm zirconium silicate rotor suitable for Doty Scientific probe. Single-shot cross-polarization contacts of 1 ms were used with matching radiofrequency field amplitudes of 75 kHz. Up to 120,000 (140,000 for light fractions) 500 W free induction decays were co-added with a delay time of 1 s and a sweep width of 20 kHz. The free induction decays were zero-filled to 4 K data points before Fourier transformation. Magic angle spinning rates were 4 kHz. Peak areas were measured with an integrator. The composition of organic matter was determined by assigning spectral regions and chemical shift limits (Arshad et al. 1988) as follows: aliphatic-C, 0– 105 ppm; aromatic-C, 105–150 ppm; phenolic-C, 150–170 ppm and carboxylic-C, 170–190 ppm. Statistical analyses Variance in soil properties among the four cropping systems was analyzed using the general linear model procedure (SAS Institute Inc. 1990). Significance was declared at P \ 0.05, and treatments were separated using the LSD test.

Results and discussion Soil structure Soil bulk density (BD) was greater in the CB and RF treatments than in either CG or CL treatment indicating less pore space in surface soil resulting from compaction by frequent tillage operations particularly under continuous barley (Table 1). As indicated in section on soil sampling and analyses, bulk density data were not collected in the initial year 1968. Bulk density has not been determined in older studies reporting SOC concentrations with different management. However, the BD can be estimated through pedotransfer functions, most typically from soil organic matter content and soil texture (Benites et al. 2007; Franzluebbers 2010). Soil C concentration is often converted to C mass per unit area by multiplying it with BD to a fixed depth. It has been demonstrated that the equivalent soil mass (ESM)

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Table 1 Bulk density, aggregate-size distribution (%) and mean weight diameter (MWD) in the 0–75 mm soil under different cropping systems in 1992 Treatment

Bulk density (Mg m-3)

Aggregate size (mm) 8–4

4–2

2–1

1–0.5

0.5–0.2

0.2–0.1

\0.13

MWD (mm) 1.48

CB

1.19

14.9

9.3

8.2

13.5

14.6

10.5

29.0

CG

1.04

25.2

13.9

10.7

9.6

9.1

5.9

25.6

2.21

CL

1.13

23.4

8.6

8.1

12.1

13.7

9.7

24.5

1.95

RF

1.22

20.4

11.4

9.1

9.8

13.5

9.5

26.3

1.85

LSD0.05

0.08

8.9

3.8

3.9

2.8

3.2

2.4

9.7

0.54

CB continuous barley, CG continuous grass, CL continuous legume, RF forage-barley rotation

correction should be used when comparing soil C stocks in genetic horizons among land use or management practices (Ellert and Bettany 1995). However, soil can vary spatially and temporally (Lee et al. 2009). Using simple simulations and field data, Lee et al. (2009) concluded that the fixed depth method is often not suitable and might be less accurate than direct C concentration measurements. Their data suggest that correcting soil C stocks for BD using the maximum or minimum ESM is not sufficient to nullify confounding effects by natural or land use or management-induced changes in BD. Neverthless, the most important factor is the uncertainty around BD measurements; when using BD measurements with great uncertainty, the best correction method is more difficult to determine. The data on aggregate-size distribution and mean weight diameter (MWD) indicate that soil structure was positively affected under CG as well as under other treatments (CL and RF) with forage crops (Table 1). The increase in MWD under CG, CL and RF treatments is mainly due to a greater proportion (ranging from 31.8 to 39.1%) of macro-aggregates (2–8 mm) with a corresponding decrease in percentages of smaller aggregates (\1 mm). Soil under continuous barley (CB) had the lowest MWD (1.48 mm) and the lowest percentages (24.2%) of water stable aggregates ([2 mm) resulting from frequent tillage used under this treatment. Franzluebbers et al. (1999) observed similar changes in soil aggregation in response to type and frequency of tillage. Their results showed a decline in MWD of water-stable aggregates in surface soil from 1.23 mm with in-row chisel to 1.12 with paraplow. In another study, planting of alfalfa in an excessively tilled bare soil (fallow) increased the MWD of aggregates from

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1.5 to 2.3 mm during a 5-year period (Angers 1992). Frequent tillage under the barley monoculture (CB treatment) brings macro-aggregates to the surface where it is then exposed to freeze-thaw cycles and subject to raindrop impact (Paustian et al. 1997) thereby increasing the susceptibility of macro-aggregates to disruption and disintegration into microaggregates. Some researchers believe that microaggregates are bound together by young organic matter into larger macro-aggregates (Jastrow et al. 1996; Six et al. 2000). Reduction in aggregation by tillage intensity results in release of labile SOM (Elliott 1986) and its increased availability for microbial decomposition (Six et al. 2000). Carbon and nitrogen in whole organic matter and its light fraction The data for whole soil and light fraction C and N (Table 2) show that: (i) all cropping systems suffered a loss of SOC (a decrease by about 19% under CG and CL, 29% under RF and 32% under CB), and light fraction C (a decrease by 13% under CG, 32% under CL, 46%under RF and 57% under CB) and light fraction N (a decrease of 13% under CG, 17% under CL, 40% under RF and 55% under CB) compared with the initial contents in 1968, (ii) the decrease was most for soil organic materials under CB followed closely by the RF treatment, and (iii) loss of C and N in soil organic materials from the CL and CG treatments were least. The decline from the initial level reflects the loss of organic matter typically associated with cultivation of soil previously under native vegetation (Tiessen and Stewart 1983). The poor results with continuous barley and, to a lesser extent, rotational barley cropping phase of rotational


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Table 2 Influence of long-term (24 years) cropping systems on bulk soil organic C and total N, and the light fraction (LF) dry matter, C and N contents of the surface soil (0–75 mm) Treatment

Light Fraction

Whole soil

Dry weight (g kg-1 soil)

C (g kg-1 soil)

N (g kg-1 soil)

C/N

C (g kg-1 soil)

N (g kg-1 soil)

C/N

Initiala

26.3

8.82

0.424

20.9

40.2

3.48

11.7

CB

15.6

3.83

0.192

20.1

27.2

2.54

10.7

CG

25.2

7.64

0.368

20.8

32.2

2.80

11.5

CL

22.3

6.01

0.350

17.3

32.5

2.90

11.2

RF

18.4

4.72

0.254

18.6

28.6

2.70

10.7

2.7

0.75

0.053

3.6

3.6

0.51

2.3

LSD0.05

CB continuous barley, CG continuous grass, CL continuous legume, RF forage-barley rotation a

Initial refers to the baseline year, 1968: other data are for the year 1992

(RF) cropping are attributed to deterioration of soil structure and decline in soil quality after many years of soil tillage and cultivation. In contrast, cropping systems with perennial forage crops were cultivated less frequently: about once every 6 years for reseeding with bromegrass and every 4–5 years with forage legumes. Variations in root mass among annual and perennial crops may have contributed to variations in SOM, however, it is likely that annual tillage operations associated with CB had more impact on SOM. Soil organic C is a dynamic pool, and net changes in storage often are informative than absolute quantities (Ellert et al. 2002). Recent estimates of soil organic C (SOC) loss suggest that current SOC reserves in surface soils in Western Canada are about 20–30% lower than those of corresponding uncultivated sites (Janzen et al. 1998; McGill et al. 1986). Using data from several studies, Davidson and Ackerman (1993) estimated a 40% SOC loss from the A horizon and about 30% loss from the entire soil solum after 20 year following cultivation, with majority of this loss occurring within the first 5 years. Our data show that the light fraction C and total SOC under CB decreased by 32 and 40%, respectively after 24 years of cropping. These findings are consistent with the data reported for other long-term studies in southern Canadian prairies, which show 38% decline in light fraction C concentration under fallow-wheat or fallow-wheatwheat rotation cropping (Janzen et al. 1992; Bremer et al. 1995). The C concentration of the light fraction, as a percentage of total SOC, was higher in CG and CL (23.7–18.5%, respectively) than in CB and RF

(14.1–16.5%, respectively) treatments (Table 2). These results reflect the negative contribution of intensive tillage operations during seed-bed preparation for barley cultivation in the CB and RF cropping systems. In cropping systems with perennial forage crops, the decline was modest for CL while the CG treatment did not show any significant change. Acid-hydrolysable monosaccharide The soil under CG contained the highest (6,300 mg kg-1) whereas the soil under CB and RF the lowest amounts of total acid-hydrolysable sugars among the treatments (Table 3). Higher concentration of carbohydrates in soils under CG likely contributed to improved soil aggregation (Angers and Mehuys 1989) under this treatment than other cropping systems studied. Concentrations of glucose, galactose and mannose in soil under CG were higher

Table 3 Monosaccharide contents (mg kg-1) in soil (0–75 mm) under continuous barley (CB), continuous grass (CG), continuous legume (CL) and rotation forage (RF) at the study site in 1992 LSD0.05

Monosaccharide

CB

CG

CL

RF

Glucose

1,560

1,880

1,740

1,480

300

450

890

610

480

280

Galactose Mannose

860

910

880

730

180

Arabinose

1,010

1,310

1,260

1,020

220

Xylose Totala

910 4,790

1,310 6,300

1,010 5,490

980 4,680

210 1,000

a

Total may not add up to sum of the sugars due to rounding

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than in any other treatment. Since the soil microbial population synthesizes predominantly these hexose sugars (Oades 1984), our soil carbohydrate data indicate greater biochemical activities in soil under CG than under CB or RF. Polysaccharide mucilages in soils derived from plant roots, bacteria and fungi are believed to be effective glues binding soil particles (Oades 1984). Microbial biomass C and N

Distribution and type of organic C The distribution and type of C in light fractions and bulk soil organic matter under different cropping systems as characterized by the 13C-NMR spectroscopy are summarized in Table 5. Among the treatments studied, the light fractions organic materials separated from soil under the CB monoculture contained the highest amounts of aromatic-C and the lowest contents of aliphatic components including

Table 4 Soil microbial biomass C and N as influenced by 24 years of croppinga Microbial C (mg kg-1soil)

Microbial N (mg kg-1soil)

Continuous barley

373

40

Continuous grass

497

54

Continuous legume

441

68

Rotation forage

398

48

LSD

167

22

a

0.05

0–75 mm depth, sampled in spring 1992

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Types of C compounds

Cropping systems CBa

CG

CL

RF

Aliphatic-C (%)

67.0 (59.8) 73.3 (65.3) 69.6 (62.4) 70.1 (63.4)

Aromatic-C (%)

21.6 (25.0) 18.2 (22.6) 18.2 (22.1) 19.2 (22.0)

Phenolic-C (%)

6.2 (8.3)

4.7 (4.7)

5.5 (5.2)

5.4 (9.2)

Carboxylic-C (%)

5.2 (6.9)

3.9 (6.4)

4.8 (10.3)

5.3 (5.4)

Aromaticitya

Soil microbial biomass C was highest under grass and lowest under continuous barley, with CL having an intermediate level of microbial C (Table 4). Soil microbial biomass N was highest under the CL cropping system and lowest under continuous barley. The C:N ratios of the microbial biomass varied from 7.5 under CL to 9.6 under CG, however, the differences were not significant (data not shown). The microbial C and N trends follow those of other soil labile C (or N) sources, e.g., the light fraction C and N, and total acid-extractable sugars. The labile C sources provide the substrates to support the soil microbial activities. Janzen et al. (1992) found a close relation between soil respiration and the light fraction C.

Cropping systems

Table 5 Composition of organic matter in the light fractions and whole soil (in brackets) under different cropping systems as characterized by 13C CP/MAS NMR

29.3 (35.8) 23.8 (29.5) 26.9 (30.4) 26.0 (33.0)

CB continuous barley, CG continuous bromegrass, CL continuous legume, RF forage-barley rotation a

AromaticCþPhenolic C Aromatic CþPhenolic CþAliphatic C

labile substances such as carbohydrates, proteins and peptides. A similar trend was observed from the 13CNMR spectra (shown in brackets of Table 5) of whole soils although here aliphatic-C was lower than in the light fraction with a concurrent increase in aromaticity (i.e., the fraction of C that is aromatic). However, aromatic-C and phenolic-C were higher in whole soils than in light fraction materials. Soils under RF treatment exhibited the highest concentration of phenolic-C. This is likely due to intensive tillage operations carried out to break the sod and prepare seed-bed for planting barley after 3 years of forage production. The light fraction isolated from the soil under CB contained the most aromatic-C among the treatments. The aromaticity ranged from 24% in light fractions from soils under CG to 29% in light fractions from the soil under CB. The CG treatment had the lowest phenolic-C and carboxylicC and the least aromaticity in the light fraction materials and bulk SOC. Of all the treatments studied, the soils under continuous grass had the highest contents of aliphatic components which included the carbohydrates previously mentioned. This may be, in part at least, responsible for reduced aromaticity in soils under continuous grass. These data indicate the positive influence of long term forage crops on soil quality. Several researchers have advocated numerous sustainable management strategies to conserve soil organic matter for building soil quality (Doran et al. 1996). A minimum data set (MDS) of indicators, as proposed by researchers (Arshad and Coen 1992; Doran and Parkin 1994; Arshad and Martin 2002), is


required to assess an improvement or deterioration in soil quality. A comprehensive approach to interpreting soil quality data has been recently developed by USDA-ARS (Andrews et al. 2004) by integration of indicator scores into a single overall health index. However, the benefits of having a single, integrated index as indicator of overall soil health remain controversial (Wolfe 2006). Soil organic matter is a relatively stable parameter that reflects the influence of management and crop type over periods of decades, and is of essential importance for soil quality (Christensen and Johnston 1997). Because of its crucial role in influencing many soil characteristics and processes that are important for soil functioning soil organic matter can be considered a suitable integrating soil parameters to evaluate changes in soil quality within a given ecological region. Our data in this study show that the key indicators such as soil structure (aggregate stability) and soil organic matter quantity and quality were negatively impacted by excessively tilled monoculture cropping system (continuous barley) but the perennial grass and legume-based crop rotations significantly improved the selected key indicators. Adoption of these soil quality enhancing practices by the landowners can result in appreciable amounts of C sequestration and significantly curtail the greenhouse gas emissions. Our data, as well as findings of other researchers (Wolfe 2006; Lal 2007; Franzluebbers 2010), demonstrate that by following the soil conserving strategies farmers can derive monetary benefits by improving the health of their soil for longterm sustainable production as well as tap into a multi-billion-dollar carbon market. The global C market in 2007 is about $30billion, but has the potential to grow to $ 1trillion by 2020 or before (Lal 2007). It should be pointed out, however, that the degree of cropping systems impact is site-specific (Franzluebbers et al. 2010) and will no doubt vary with climatic regions.

Conclusion Our data showed that cropping systems that include grass and/or legume forage crops had better soil structure (i.e., more stable aggregates) than soil under continuous barley. Soil under continuous bromegrass (CG) and continuous forage legume (CL) were higher

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in soil and light fraction organic matter than the other two treatments. Rotation cropping that alternate 3 years of barley production with 3 years of forage production had intermediate levels of soil structure and organic matter content. Soil under CG had the highest and under continuous barley (CB) the lowest amounts of acid-hydrolysable sugars amongst the treatments. Microbial biomass C and N tend to follow the trend among treatments in whole and light fraction organic matter. Soil under CG and CL and the barley/forage rotation had the higher amounts of aliphatic-C (indicating enrichment in carbohydrate, proteins and peptides, thus improving organic matter quality) than soil under CB. These results indicate the positive contribution of forage crops to sequester carbon and improve soil quality. Acknowledgments This project was supported by the National Soil Conservation program (NSCP). The assistance of R. Azooz and A.U. Haq is gratefully acknowledged.

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