2. 46 Mha (Zalles et al., 2019). The study reports that circa 80 % of the
new cropland areas were converted from grazing lands. Nonetheless,
the remaining 20 % of the expansion on cropland area was due to
conversion of lands under natural vegetation, notably over savannas
and Cerrado woodlands in Central Brazil but also over humid tropical
forests in the Amazon region (Zalles et al., 2019).
Early adoption of conservation agriculture (CA) practices could
hypothetically mitigate or prevent SOC losses in these new cropland
areas. Conservation agriculture is a major global initiative promoted to
recover SOC in degraded agricultural soils by using minimum soil dis-
turbance, permanent soil cover and crop rotation (Jat et al., 2014; Lal,
2016; Minasny et al., 2017). No-till (NT) is considered a key component
of CA, providing minimum soil disturbance by planting directly through
soil cover. No-till was already shown to partially offset SOC losses from
agricultural soils following conversion of natural ecosystems (Kopittke
et al., 2017; Luo et al., 2010). However, recent studies showed that the
association of NT with best crop management practices could increase
crop yields and C inputs to the soil, thus augmenting SOC accrual in NT
soils up to the levels of natural soils (de Oliveira Ferreira et al., 2018b,
2016; Nicoloso et al., 2020).
Soil organic carbon accrual in NT soils is favoured by fertilization
(de Oliveira Ferreira et al., 2018a; Kirkby et al., 2014; Manzoni et al.,
2012; Poffenbarger et al., 2017; Van Groenigen et al., 2017), ameli-
oration of subsoil acidity (Dalla Nora et al., 2017a; de Oliveira Ferreira
et al., 2018a), use of irrigation, high yield cultivars and increased plant
density (Follett et al., 2013; Grassini et al., 2011; Grassini and Cassman,
2012; Schwalbert et al., 2018; Stewart et al., 2017), use of crop rotation
and intensification of cropping systems (de Oliveira Ferreira et al.,
2018a, 2016; Nicoloso et al., 2020; Pittelkow et al., 2014) and the use
of organic amendments providing external C inputs (Nicoloso et al.,
2018, 2016; Poulton et al., 2018; Powlson et al., 2012; Xia et al., 2017),
In this sense, the use of animal manure as organic fertilizers (e.g.
swine slurry, cattle manure and poultry litter) is particularly interesting
for agricultural regions where these sources of nutrients are abundant
and readily available such as in Southern Brazil (Aita et al., 2014; Kunz
et al., 2009). The use of swine slurry in substitution or complementarily
to mineral fertilizers was already shown to increase SOC in a Red Oxisol
under NT from Southern Brazil in comparison with mineral fertilizers
alone (Mafra et al., 2014). However, other studies showed that the use
of composted manure may yield greater recovery and prolonged storage
of external C inputs as SOC than noncomposted manure (Lynch et al.,
2006; Nicoloso et al., 2018, 2016). Composted organic sources are
known to be thermodynamically resistant to decomposition (Huang
et al., 2006), promoting slower decomposition rates (Grave et al.,
2015), higher soil aggregation and SOC protection within aggregates
(Nicoloso et al., 2018).
The objective of our study was to test the hypothesis that early
adoption of NT associated with organic fertilization could mitigate SOC
losses from a grassland following its conversion to cropland when the
natural vegetation was suppressed and the soil was disrupted for
amelioration of soil acidity with lime incorporation. Thus, we assessed
short-term temporal changes (5 years) on TOC and TN stocks, soil C and
N pools within sand and silt + clay fractions and soil C and N quality
indices in a Nitisol from Southern Brazil under conventional tillage (CT)
and NT system. The area was cultivated with maize and black oats and
amended with mineral (urea) and organic N sources (pig slurry, di-
gested pig slurry and composted pig slurry). We also evaluated TOC and
TN concentrations within soil aggregates to infer on the mechanisms
regulating SOC stabilization and depletion according to soil tillage
systems and contrasting N sources.
2. Material and methods
2.1. Experimental site
This study was initiated in a natural grassland area located in
Concordia-SC, Brazil (27º18'53”S, 51º59'25"O). The local climate was
humid subtropical (Cfa), with mean annual temperature and pre-
cipitation of 18 °C and 1.800 mm (Wrege et al., 2012). The soil was
classified as a Rhodic Nitisol (IUSS Working Group WRB, 2015). The
soil was sampled (0−10 cm) in March/2012 and analyzed using stan-
dard methods (Embrapa, 2011). The soil had a loamy texture with 250,
460, and 290 g kg−1
of clay, silt, and sand, respectively, and the initial
chemical properties were: pH-H2O(1:1) 5.3, Al3+
0.3 cmolc dm-3
,
PMehlich-1 6.6 mg dm-3
, KMehlich-1 249.6 mg dm-3
, Ca 7.5 cmolc dm-3
, Mg
3.3 cmolc dm-3
, CEC 11.9 cmolc dm-3
, and base saturation of 68 %.
2.2. Conversion of grassland to cropland
The four main corners of the experimental area were permanently
marked with posts and the grassland vegetation was suppressed with
glyphosate (2.4 kg ha−1
) in Mach/2012. In April/2012, we applied 2
Mg ha−1
of lime for amelioration of soil acidity (SBCS-CQFS, 2004).
Lime was incorporated with one pass of a disk plow equipped with a set
of three disks with 26 in. of diameter (Baldan ARH(L), Matão, Brazil)
followed by two passes of a tandem disk harrow equipped with a set of
20 disks with 20 in. of diameter (Baldan HI, Matão, Brazil). The
plowing and disking operations were performed to a 20–25 and 8−10
cm depth, respectively. The area was sown with 80 kg seeds ha−1
of
black oats (Avena strigosa Scherb.) with 20 cm row spacing using a no-
till drill (Eickhoff ESG-302, Três de Maio, Brazil) equipped with offset
double disk openers working to a 3−5 cm depth. In September/2012,
the black oats were killed at the flowering stage with glyphosate (1.2 kg
ha−1
) and the residues were maintained on the soil surface.
2.3. Treatments and experimental design
The experiment was established in September/2012 using a double-
cropping system with maize (Zea mays L.) as a summer grain crop and
black oats as a winter cover crop. Maize was planted between early-
September and mid-October, and the black oats were sown between
mid-March to mid-April, depending on weather and crop development
in each year. Plots measuring 10 × 25 m (Length x Width) were ar-
ranged in a completely randomized blocks design with four replica-
tions, where two tillage systems were tested: conventional tillage (CT)
and no-till (NT). The CT consisted of plowing and disking before
planting maize, and disking before sowing the black oats. Plowing and
disking operations were performed as already described. The NT con-
sisted of planting maize and sowing the black oats though crop residues
without previous soil tillage. After the tillage operations were per-
formed for CT, the both CT and NT plots were splitted in subplots
measuring 10 × 5 m (LxW) where different mineral and organic N
sources where tested: urea (UR), pig slurry (PS), digested pig slurry
(DS), composted pig slurry (CS), besides a control (CTR) treatment
without fertilization.
The organic N sources were obtained from an experimental fat-
tening pig farm where the PS was stored in anaerobic pits (Sardá et al.,
2018), the DS was treated in a upflow anaerobic sludge blanket (UASB)
biodigester (Kunz et al., 2009), and the CS was co-composted with a
mixture of wood shavings and sawdust (Angnes et al., 2013). The or-
ganic N sources were sampled and analyzed for physical-chemical
characterization (Table 1) using standard methods (APHA, 1998). The
mineral and organic fertilizers were broadcasted on the soil surface at
140 kg ha−1
of total N before planting maize in order to achieve an
expected grain yield of 9 Mg ha−1
(SBCS-CQFS, 2004). Triple super-
phosphate and potassium chloride were applied as needed to supply at
least 115 kg P2O5 ha−1
and 75 kg K2O ha−1
for fertilized plots (SBCS-
CQFS, 2004). For CT treatments, fertilizers were incorporated into the
soil with one pass of tandem disk harrow. Maize was planted at
60.000–65.000 plants ha−1
with 80 cm row spacing using no-till
planters (Eickhoff ESG-302, Três de Maio, Brazil or SB Máquinas PHPE-
4C, Cambé, Brazil) equipped with flat disk coulters to cut though crop
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
2
3. residues, shanks (10−12 cm depth) and double disk for seeds (5−8 cm
depth). The black oats did not receive fertilization. Every year, the
experimental area was harvested using a commercial combine and the
black oats were killed using glyphosate. Both maize and black oat re-
sidues were kept at the soil surface. Other farming practices followed
regional recommendations for both maize and black oats crops.
2.4. Aboveground carbon inputs
Maize and black oats were sampled in all subplots and in every year
for determination of maize stover and black oats aboveground biomass
inputs to the soil. Briefly, four plants of maize at physiological maturity
were randomly selected and cut at the soil surface. Grains were sepa-
rated and both grains and stover were oven-dried at 65 °C until constant
weight. We then calculated the harvest index (HI) for maize with HI(%)
=(G/(G + S))x100, where G and S are the grain and stover masses of
the samples, respectively, in kg. Grain yield was assessed before har-
vesting by sampling the cobs from maize plants found within two linear
meters of three different rows selected randomly inside each sub-plot.
The total area sampled was 4.8 m2
per sub-plot (9.6 % of sub-plot area).
Grains were separated, weighed and dried at 65 °C until constant
weight. The maize stover biomass was then calculated considering the
HI determined at physiological maturity and grain yield measured at
harvest. The black oats were sampled at flowering stage by cutting the
plants found inside a 0.25 m2
area using a square metal frame. Samples
were oven-dried at 65 °C until constant weight. Sub-samples of the
maize stover, black oats as well as the organic fertilizers used in this
study were finely ground and analyzed for C and N by dry combustion
(Flash 2000 CHNS-O, Thermo Scientific, Waltham, United States).
Aboveground C inputs were calculated considering the maize stover
and black oats biomass yields, the application rate of organic fertilizers
and their C contents.
2.5. Soil sampling and analysis
The soil under the grassland was sampled using a hydraulic probe
with 5-cm of diameter in March/2012. Three undisturbed soil cores
were taken from a 70 cm depth within the same area where the main
plots were establish later that year (September/2012). This procedure
was used to establish a pretreatment baseline for the main plots with
the CT and NT treatments. Soil cores that were compacted or disturbed
during sampling were discarded. The soil cores were separated into
seven depth increments (0−5, 5−10, 10−20, 20−30, 30–40, 40−60
and 60−70 cm) with attention to prevent cross-contamination between
sampling depths. Soil cores were composited by plot and depth,
weighed and stored at 4 °C. A subsample was taken for determination of
soil moisture by oven-drying at 105 °C until constant weight. The soil
bulk density was calculated using the mass of dry soil per depth, core
diameter and number of cores taken per plot. The remaining sample
was air-dried and passed through a 2-mm sieve. All visible plant roots
and residues were removed during the sieving process. A subsample
was ground with a mortar and pestle to pass through a 500 μm sieve,
analyzed for C and N by dry combustion (Flash 2000 CHNS-O, Thermo
Scientific, Waltham, United States), and used for determination of TOC
and TN stocks. Other subsamples of the 2-mm sieved soil from the 0−5,
5−10, 10−20 and 20−30 cm layers were used for separation of sand
(> 53 μm) and silt + clay (< 53 μm) fractions by wet sieving
(Cambardella and Elliott, 1992). Both fractions were recovered,
weighed and analyzed for C and N by dry combustion. The particulate
organic C (POC) and N (PN) was determined within the sand fraction,
and the mineral-associated organic C (MAOC) and N (MAN) corre-
sponded to the silt + clay fraction. In March/2017, the soil was sam-
pled again by pulling two undisturbed soil cores from each sub-plot
using the procedures and analysis already described.
2.6. Soil organic carbon and nitrogen stocks
Soil TOC and TN stocks were calculated using the soil bulk density
and soil C and N concentrations by depth for samples collected in 2012
and 2017. Soil POC, PN, MAOC and MAN stocks were then calculated
also considering the mass of both sand and silt + clay fractions and
their concentrations of C and N. Soil C and N stocks as measured in
Table 1
Application rate and characteristics of the organic fertilizers used in this study.
Material Year Characteristics Application rate
DM VS TOC TN C/N Org-N NH4-N NO3-N Min-N P K
– % – —————————— kg m−3
——————————
—————————— kg m−3
——————————
– % – ———— kg m−3
————
– m3
ha−1
–
PS1
2012 6.0 4.6 29.0 4.4 6.6 1.7 2.7 n/d 62.0 0.7 2.1 31.7
2013 n/d n/d 15.6 4.1 3.8 0.9 3.2 n/d 78.4 1.2 1.5 34.1
2014 2.6 1.7 9.3 3.0 3.1 1.0 2.0 n/d 67.4 0.7 1.7 46.9
2015 13.3 11.2 51.5 5.7 9.1 2.4 3.3 n/d 58.2 2.1 2.0 24.7
2016 1.8 1.2 6.1 2.4 2.6 0.8 1.6 n/d 65.6 0.3 1.0 59.1
DS1
2012 7.0 3.8 17.7 5.2 3.4 2.5 2.6 n/d 50.7 3.2 0.9 27.1
2013 n/d n/d 6.3 2.6 2.5 0.5 2.1 n/d 81.1 0.2 1.2 54.8
2014 1.3 0.8 4.3 1.9 2.3 0.5 1.3 n/d 71.5 0.4 0.9 75.6
2015 1.2 0.7 4.0 1.9 2.2 0.3 1.6 n/d 82.7 0.2 1.0 74.5
2016 0.8 0.4 1.9 1.8 1.1 0.2 1.6 n/d 88.7 0.1 0.7 78.7
– % – —————————— g kg−1
——————————
—————————— g kg−1
——————————
– % – ———— g kg−1
————
- Mg ha−1
-
CS2
2012 29.1 n/d 317.0 16.6 19.1 15.1 1.2 0.3 8.9 10.9 18.9 29.0
2013 47.3 n/d 249.6 23.6 10.6 23.5 0.1 0.0 0.5 22.6 9.6 12.5
2014 43.8 n/d 378.0 21.6 17.5 19.8 0.8 1.0 8.5 10.5 11.1 14.8
2015 42.0 n/d 325.1 18.3 17.8 18.2 0.1 0.0 0.3 16.5 9.4 18.3
2016 30.0 n/d 299.0 17.5 17.1 17.0 0.0 0.5 3.1 12.5 10.9 26.7
PS: pig slurry; DS: digested pig slurry; CS: composted pig slurry; DM: dry matter; VS: volatile solids; TOC: total organic carbon; TN: total nitrogen; C/N: total organic
carbon/total nitrogen ratio; Org-N: organic nitrogen; NH4-N: ammonium-nitrogen; NO3-N: nitrate-nitrogen; Mineral-N: mineral nitrogen as a percentage of TN; P:
phosphorus; K: potassium; n/d: not determined.
1
Results are expressed on a fresh matter basis.
2
Results are expressed on a dry matter basis.
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
3
4. different pools were then corrected for differences in soil bulk density
and compared using equivalent soil masses (ESM) following the method
described by Wendt and Hauser (2013). The procedures used for cal-
culation soil C and N stocks in ESM corresponded to the original
method described by Ellert and Bettany (1995), but using cubic spline
functions obtained from the SRS1 Cubic Spline for Excel™ add-in (SRS1
Software, Boston, United States). The use of cubic spline functions
eliminates laborious calculation and possible errors in the comparison
of treatments with multiple sampling times and reference soil masses,
and provide a better fit to changes in SOC concentrations across all
sampling depths than methods based on linear interpolation (Wendt
and Hauser, 2013). Briefly, we used piecewise series of cubic poly-
nomial curves to calculate soil C and N stocks in the cumulative ESM of
0−500, 0–1,100, 0–2,400, 0–3,700, 0−5,000 and 0–7,500 Mg ha−1
,
which corresponded approximately to the average soil masses mea-
sured for the 0−5, 0−10, 0−20, 0−30, 0–40 and 0−60 soil depths as
sampled in 2012. The SOC concentration and soil mass measured in the
60−70 cm soil depth were used only for correction of SOC stocks in
ESM, and the results were reported only to 0–7500 Mg ha–1
or 0–60 cm
depth. Soil C and N stocks in the intermediate ESM of 500–1,100,
1100–2,400, 2400–3,700, 3700−5,000 and 5000–7,500 Mg ha−1
were
calculated by subtraction and corresponded approximately to the
5−10, 10−20, 20−30, 30–40 and 40−60 cm depths. Although soil C
and N stocks as measured in different pools were compared in ESM, the
results were expressed as soil depths for ease of interpretation. Changes
on soil C and N stocks were calculated in reference to the original stocks
as measured in March/2012 in the soil under grassland. This procedure
accounted for temporal changes of SOC within treatments as well for
the spatial variability of the experimental area (Olson et al., 2014).
2.7. Soil organic C and N quality indices
The concentrations of TOC, POC, TN and PN measured in the 0−5
and 5−30 cm soil layers were used to calculate the their respective
stratification ratios (CSR, POCSR, NSR and PNSR) (Franzluebbers,
2002; Zanatta et al., 2019) with:
SR = C or N (g kg−1
, 0−5 cm) / C or N (g kg−1
, 5−30 cm) (1)
here, SR is the CSR, POCSR, NSR or PNSR calculated as the ratio of the
C or N concentrations of the respective pool in the 0−5 cm soil layer
and the concentration of the same C or N pool in the underlying 5−30
cm soil layer.
We also calculated the carbon and nitrogen pool index (CPI and NPI,
respectively), the carbon and nitrogen lability index (CLI and NLI, re-
spectively) and the carbon and nitrogen management index (CMI and
NMI, respectively) as proposed by Blair et al. (1995) and revised by
Chatterjee et al. (2018) and Zanatta et al. (2019):
PI = TOCtrat or TNtrat (g kg−1
, 0−30 cm) / TOCref or TNref (g kg−1
,
0−30 cm) (2)
LI = CLtrat or NLtrat / CLref or NLref (3)
CL/NLtrat/ref = POC or PN (Mg ha−1
, 0−30 cm) / MAOC or MAN (Mg
ha -1
, 0−30 cm) (4)
MItrat/grass = PItrat/grass x LItrat/grass (5)
where, PI is the CPI or NPI calculated as the ratio of the concentrations
of TOC or TN in the 0−30 cm soil layer of a given treatment and the
concentrations of TOC or TN in the 0−30 cm soil layer of the grassland
soil used as reference for comparison; LI is the carbon or nitrogen la-
bility (CL and NL respectively) in a given treatment as a ratio of the
grassland soil used as reference of comparison; CL and NL area calcu-
lated as a ratio of the POC or PN stocks in the 0−30 cm soil layer of a
given treatment or the grassland soil and the respective MAOC and
MAN stocks in the same layer of a given treatment or the grassland soil;
and MI is the CMI or NMI calculated as the product of the PI and LI for a
given treatment or the grassland soil.
2.8. Aggregate-size distribution
In March/2017, undisturbed soil samples (20 × 20 cm) were taken
from the 0−5 cm soil layer of selected treatments: CT CTR, CT UR, CT
CS, NT CTR, NT UR, and NT CS. Samples were passed through an 8-mm
sieve removing large roots and plant residues, air-dried and stored at
room temperature in rigid containers until analysis. Three replicates
with 40 g of air dried samples were wet sieved for assessment of soil
aggregates stability following the procedure described by Gulde et al.
(2008). Briefly, each replicate was evenly distributed over a 2000 μm
sieve previously mounted in a wet-sieving machine (Tecnal TE-3300,
Piracicaba, Brazil) equipped with 10 l plastic buckets. The wet-sieving
machine oscillated vertically the sieves with a stroke length of 4 cm at
0.5 Hz. The sieve was set to its lowest vertical position and the buckets
were slowly filled with water until covering the samples, which were let
slake for 5 min. Root and plant residues larger than 2 mm that floated
were removed and oven-dried at 65 °C until constant weight for cor-
rection of initial sample mass. Water-stables aggregates (WSA) were
then separated by wet-sieving during 2 min, when the WSA remaining
on the top of the sieve were collected in an aluminum pan. The soil and
water collected in the bucket was poured in a 250 μm sieve mounted in
the wet-sieving machine set with another clean bucket. The sample was
wet-sieve for another 2 min. The same process was repeated using a 53
μm sieve. The sampled that passed through the last sieve was let decant
for 24−48 h, excess water was poured off, and the decanted soil was
collected in an aluminum pan. Samples with the four WSA size frac-
tions, named large macroaggregates (> 2000 μm), macroaggregates
(250–2,000 μm), microaggregates (53−250 μm), and silt + clay frac-
tion (< 53 μm) were oven-dried at 65 °C until constant weight. Sub-
samples were ground with mortar and pestle and analyzed for C and N
by dry combustion. Other sub-samples from WSA fractions > 53 μm
were separated for sand correction (Elliott et al., 1991). Briefly, samples
with 2−4 g of WSA were oven-dried at 105 °C until constant weight. A
fivefold volume of 5 g L−1
sodium hexametaphosphate was added to
samples, which were left overnight and then shaken at 350 rpm for 4 h.
The dispersed soil retained on a 53 μm sieve was oven-dried at 105 °C
until constant weight.
2.9. Statistical analysis
The annual aboveground C inputs from maize stover, black oats, and
the cumulative C inputs from maize, black oats and organic fertilizers
were assessed using analysis of variance (ANOVA) with SAS PROC
MIXED (SAS 9.4, SAS® Institute Inc., Cary, NC, USA). The same pro-
cedure was used for the soil variables assessed in this study. All analysis
were performed by soil layer. We used a split-plot design with rando-
mized blocks, assessing the effects of soil tillage in the main plots, N
sources as subplots and the interactions of tillage and N sources. We
included the WSA size classes as a variable of the model to test its ef-
fects and interactions with soil tillage and N sources over the mass of
WSA and the C and N content within WSA. When the F test was sig-
nificant (p ≤ 0.05), we used the Tukey-Kramer test to assess differences
between soil tillage systems, N sources and their interactions for 2017
data. We also used the t-test to assess differences promoted by tillage
and N sources treatments as measured in 2017 and compared with the
grassland as sampled in 2012. Correlations between soil C and N quality
indices, WSA and TOC and TN stocks were assessed using the Pearson’s
correlation test. All statistical comparisons were made at α = 0.05
probability level, unless noted otherwise.
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
4
5. 3. Results
3.1. Aboveground C inputs
Annual C inputs from maize stover biomass increased from 4.5 Mg C
ha−1
yr−1
in the CTR treatment to 5.4–5.7 Mg C ha−1
yr−1
with the
application of 140 kg total N ha−1
yr−1
as either UR, PS or DS
(Table 2). The application of CS promoted intermediary C inputs from
maize stover (4.9 Mg C ha−1
yr−1
), not differing from CTR and UR. No
significant differences on maize C inputs were noticed between soil
tillage systems (Table S1). Carbon inputs from black oats were not af-
fected by either tillage systems or N sources, averaging 1.8 Mg C ha−1
yr−1
. Total C inputs to the soil included maize stover, black oats bio-
mass as well the organic fertilizers used in this study. Again, no dif-
ferences were noticed for tillage systems. Nonetheless, soils amended
with CS had 2.3 Mg C ha−1
yr−1
of external C inputs, totalizing 8.9 Mg
C ha−1
yr−1
. The application of PS and DS added 0.7 and 0.3 Mg C
ha−1
yr−1
to the soil, with respective total input of 8.1 and 7.9 Mg C
ha−1
yr−1
. Treatments with mineral and no fertilization (UR and CTR,
respectively) had the lowest total C inputs of 7.1 and 6.2 Mg C ha−1
yr−1
, differing significantly from soils amended with organic fertilizers,
such as PS and CS.
3.2. Total soil C and N stocks
Nitrogen sources had no significant effect on total C and N stocks for
any of the soil layers (Table S1). Nonetheless, NT had higher TOC and
TN stocks in the 0−5, 5−10, and the cumulative 0−30 and 0−60 cm
soil layers as compared with the CT soils, on the average of N source
treatments in 2017 (Fig. 1). No differences on TOC and TN stocks were
noticed between tillage systems for other soil layers. No-till increased
TOC stocks in the soil surface (0−5 cm) and consequently in the cu-
mulative 0−30 cm layer (+2.9 Mg TOC ha−1
) as compared with the
original stocks under the grassland. In contrast, significant losses of
TOC were observed in the CT soil in the 0−5 and 5−15 cm layers,
which were cumulative to the 0−30 cm soil layer (-4.0 Mg TOC ha−1
).
Nonetheless, significant TOC losses were observed in deeper soil layers
under both CT (30–40 and 40−60 cm) and NT (40−60 cm) as com-
pared with the grassland. Thus, TOC stocks in NT soils did not differed
from the original stocks under grassland when sampled 60 cm depth. In
contrast, TOC losses under CT soils increased to 11.5 Mg TOC ha−1
in
the overall soil profile (0−60 cm) as compared with the grassland soil.
Both CT and NT soils had significant losses of TN in comparison with
the original stocks under grassland throughout the soil profile, with the
exception of the 0−5 and 10−20 cm soil layers of NT soils. No-till soils
have lost 0.6 and 1.7 Mg TN ha−1
in the respectively 0−30 and 0−60
cm soil layers. Greater losses were observed under CT with a decrease of
1.3 and 2.6 Mg TN ha−1
in the same soil layers, respectively.
3.3. Particulate and mineral associated C and N fractions
No-till soils had higher POC and PN than CT soils in the 0−5, 5−15
and the cumulative 0−30 cm soil layer as measured in 2017 (Fig. 2).
The NT soils showed increased POC and PN in the 0−5, 5−10 and
10−20 cm soil layers as compared with the original pools under the
grassland. In contrast, CT soils showed significant losses of POC in the
soil surface (0−5 cm) and PN in the 0−5 and 5−10 cm soil layers.
Nonetheless, both POC and PN increased in the 10−20 cm soil layers
under CT as compared with the grassland. Both CT and NT soils had
increased POC (1.7 and 7.5 Mg POC ha−1
, respectively) and PN (95 and
580 kg PN ha−1
, respectively) in the cumulative 0−30 cm soil layer as
compared with the grassland.
Soil tillage systems had limited effect over the MAOC and MAN
fractions as measured in 2017, when NT soils had greater MAOC and
MAN than CT only at the soil surface (0−5 cm) (Fig. 3). Nonetheless,
when compared with the original stocks under the grassland, CT soils
had significant losses of MAOC in the 0−5, 5−10 and in the cumula-
tive 0−30 cm layer (−2.1 Mg MAOC ha−1
). No-till soils also had losses
of MAOC in the 5−10 cm layer, not differing from the grassland else-
where. Both CT and NT soils had decreased MAN in the 0−5 and 5−10
cm layers as compared with the grassland. Similarly, both CT and NT
soils had significant MAN losses in the cumulative 0−30 cm soil layer
with respective −550 and −520 kg MAN ha−1
. The nitrogen sources
tested in this study did not affect none of the POC, PN, MAOC and MAN
fractions measured in any of the soil layers (Table S1). However, the use
of CS seemed to increase POC at the soil surface layer (0−5 cm) in
comparison with other treatments at p = 0.063 (data not show).
3.4. Soil C and N quality indices
The SR of TOC, POC, TN and PN were affect by both tillage and N
sources in contrast to other soil C and N quality indices assessed in this
study were only sensitive to soil tillage systems (Table S1). Nonetheless,
NT consistently showed better soil C and N quality indices than soils
under CT (Table 3). No-till soils also presented higher C and N quality
indices than the grassland soil with the exception of the CPI (1.03) and
the NPI (0.91) indices that did not differ and decreased, respectively, in
comparison with the grassland. In contrast, CT soils had lower C and N
quality indices than observed for the grassland, with the exception of
CLI and NLI (1.12 and 1.16, respectively) that increased and the CMI
Table 2
Aboveground C inputs to a Nitisol from Southern Brazil as affected by soil tillage system and N sources on the average of the experimental period (2012-2017).
Source Tillage N source Mean
CTR UR PS DS CS
———————————————————————— Mg C ha−1
yr−1
————————————————————————
Fertilizers CT/NT 0.0 0.0 0.7 0.3 2.3 n/d
Maize CT 4.9 5.5 5.7 5.7 5.1 5.4ns
NT 4.2 5.3 5.5 5.7 4.6 5.1
Mean 4.5c1
5.4ab 5.6a 5.7a 4.9bc 5.2
Black oats CT 1.7 1.7 1.8 2.0 1.7 1.8ns
NT 1.6 1.8 1.8 1.8 1.8 1.8
Mean 1.7ns 1.7 1.8 1.9 1.8 1.8
Total CT 6.6 7.2 8.2 8.0 9.1 7.8ns
NT 5.8 7.1 8.1 7.8 8.7 7.5
Mean 6.2d 7.1c 8.1ab 7.9bc 8.9a 7.6
CT: conventional tillage; NT: no-till; CTR: control without fertilization; UR: urea; PS: pig slurry; DS: digested pig slurry; CS: composted pig slurry; ns: not significant
by F test;
1
Means followed by different letters are significantly different by the Tukey-Kramer test (p < 0.05).
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
5
6. (1.02) that did no differ from the grassland soil. Soils amended with CS
had the higher CSR, POCSR, NSR and PNSR (1.91, 1.98, 1.81 and 2.19,
respectively) than other treatments, with DS having intermediary va-
lues of CSR and NSR. Indices of CSR and POCSR also differed between
soils amended with CS and soil under grassland. The correlations of soil
C and N quality indices with TOC stocks measured in the 0−30 cm soil
layer of tillage and N sources treatments were significant for POCSR,
CPI, CMI, NPI, NLI and NMI. Significant correlations of CPI, CMI, NPI,
NLI and NMI were also observed for TN stocks for the same soil layer.
Fig. 1. Total organic C (A) and N (B) stocks in the 0-60 cm soil depth of a Nitisol from Southern Brazil under natural grassland vegetation in 2012 and following 5
years of its conversion to agriculture under continuous conventional tillage (CT) and no-till (NT). Means followed by different letters are significantly different by the
Tukey-Kramer test (P < 0.05) on the comparison of tillage systems in 2017. Horizontal error bars indicate the means’ 95 % confidence interval (95 %C.I.). The 95
%C.I. for the grassland soil was omitted for clarity. If the means’ 95 %C.I. does not bracket zero or the grassland, differences between the original stocks under the
grassland soil in 2012 and the stocks under CT or NT soils in 2017 are significant by the t-test (p < 0.05).
Fig. 2. Particulate organic C (A) and N (B) stocks in the 0-30 cm soil depth of a Nitisol from Southern Brazil under natural grassland vegetation in 2012 and following
5 years of its conversion to agriculture under continuous conventional tillage (CT) and no-till (NT). Means followed by different letters are significantly different by
the Tukey-Kramer test (P < 0.05) on the comparison of tillage systems in 2017. Horizontal error bars indicate the means’ 95 % confidence interval (95 %C.I.). The 95
%C.I. for the grassland soil was omitted for clarity. If the means’ 95 %C.I. does not bracket zero or the grassland, differences between the original stocks under the
grassland soil in 2012 and the stocks under CT or NT soils in 2017 are significant by the t-test (p < 0.05).
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
6
7. 3.5. Soil C and N within water-stable aggregates
Soil tillage systems had significant interactions with WSA classes on
the distribution of the masses of WSA (Table S2). In contrast, N sources
had no effect or interactions with other factors affecting the distribution
of the masses of WSA. Also no differences were noticed on sample re-
covery within WSA fractions regarding the treatments tested in this
study, averaging 97.5 % of the original sample (Table 4). Macro-
aggregates with 250–2,000 μm accounted for 54 % of the WSA mass
(355.5 g kg−1
soil) in CT soils, followed by large macroaggregates (25
%) and the microaggregate and silt + clay classes (21 %). No-till soils
had a greater proportion of large macroaggregates with > 2.000 μm (58
% or 319.1 g kg−1
soil), with the remaining 33 % of the mass of WSA
found within macroaggregates (33 %) and the microaggregate and silt
+ clay classes (9 %). NT soils had twice the proportion of large mac-
roaggregates than CT soils with an inverse relationship found for the
macroaggregates fractions. No differences between soil tillage systems
were observed for the microaggregates or silt + clay fractions.
We found significant interactions for both tillage systems and N
sources with WSA size classes on the contents of C and N associated
with WSA (Table S2). Soil C content within WSA fractions ranged from
0.8 to 1.0 g C kg−1
soil within the silt + clay fraction (< 53 μm),
2.9–4.2 g C kg−1
soil within microaggregates (53−250 μm), 11.2–13.2
g C kg−1
soil within macroaggregates (250–2,000 μm) and 6.4–21.5 g C
kg−1
soil within large macroaggregates > 2000 μm (Fig. 4). Soil N
content within WSA followed a similar pattern ranging from 0.08 to 0.1
g N kg−1
soil within the silt + clay fraction, 0.24 to 0.32 g N kg−1
soil
within microaggregates, 0.91–1.10 g N kg−1
soil within macro-
aggregates and 0.59–1.82 g N kg−1
within large macroaggregates.
Differences between tillage systems and N sources were noticed for
storage of C and N were found only for the large macroaggregates
fraction (NT > CT and CS > UR = CTR). In CT soils, most of the C and
N were found within macroaggregates with 250–2,000 μm (54 and 52
%, respectively). The remaining C and N in CT soils were found within
large macroaggregates (26 and 28 %, respectively) and the micro-
aggregates and silt + clay fractions (20 %). In contrast, NT soils had
Fig. 3. Mineral associated organic C (A) and N (B) stocks in the 0-30 cm soil depth of a Nitisol from Southern Brazil under natural grassland vegetation in 2012 and
following 5 years of its conversion to agriculture under continuous conventional tillage (CT) and no-till (NT). Means followed by different letters are significantly
different by the Tukey-Kramer test (P < 0.05) on the comparison of tillage systems in 2017. Horizontal error bars indicate the means’ 95 % confidence interval (95
%C.I.). The 95 %C.I. for the grassland soil was omitted for clarity. If the means’ 95 %C.I. does not bracket zero or the grassland, differences between the original
stocks under the grassland soil in 2012 and the stocks under CT or NT soils in 2017 are significant by the t-test (p < 0.05).
Table 3
Soil organic C and N quality indices of a Nitisol from Southern Brazil as affected by five years of continuous conventional tillage (CT) and no-till (NT) amended with
urea (UR), pig slurry (PS), digested pig slurry (DS), composted swine slurry (CS), besides a control treatment without N (CTR).
Soil C and N indices Soil layer Grassland Tillage N source
cm CT NT CTR UR PS DS CS
TOC Stratification Ratio (TOCSR) 0−5/5−30 1.62 1.36B1,†
1.95A†
1.59b 1.56b 1.58b 1.61ab 1.91a†
POC Stratification Ratio (POCSR) 0−5/5−30 1.59 1.27B†
1.82A†
1.48b 1.42b 1.40b 1.45b 1.98a†
C Pool Index (CPI) 0−30 1.00 0.91B†
1.03A 0.95ns 0.98 1.00 0.96 0.95
C Lability Index (CLI) 0−30 1.00 1.12B†
1.39A†
1.28ns 1.20 1.15 1.22 1.42
C Management Index (CMI) 0−30 1.00 1.02B 1.43A†
1.22ns 1.19 1.16 1.19 1.37
TN Stratification Ratio (TNSR) 0−5/5−30 1.56 1.32B†
1.82A†
1.49b 1.49b 1.52b 1.54ab 1.81a
PN Stratification Ratio (PNSR) 0−5/5−30 1.70 1.27B†
1.99A†
1.57b 1.41b 1.48b 1.50b 2.19a
N Pool Index (NPI) 0−30 1.00 0.79B†
0.91A†
0.84ns 0.86 0.89 0.85 0.81
N Lability Index (NLI) 0−30 1.00 1.16B†
1.53A†
1.37ns 1.29 1.24 1.31 1.52
N Management Index (NMI) 0−30 1.00 0.91B†
1.40A†
1.18ns 1.12 1.11 1.12 1.25
1
Means followed by different uppercase letters comparing tillage systems and lowercase letters comparing N sources in the same line are significantly different by
the Tukey-Kramer test (p < 0.05).
†
significantly different from the grassland soil by the t-test (p < 0.05).
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
7
8. most of the C and N protected within large macroaggregates (59 and 60
%, respectively), followed by macroaggregates (31 and 30 %, respec-
tively) and the remaining C and N found within microaggregates and
the silt + clay fraction (10 %). The use of CS increased the proportion
of C and N protected by large macroaggregates (52 %) than by mac-
roaggregates (34 %) and the other fractions (14 %). Soils amended with
PS and without N (CTR) had no differences on C and N contents found
within large macroaggregates and macroaggregates fractions, which
accounted for 85−57 % of the C and N mass within WSA.
4. Discussion
4.1. Total soil C and N stocks
Changes on soil TOC and TN stocks over time due to agricultural
practices are relatively small in comparison with natural soil variability
(Blair et al., 1995; Olson et al., 2014). Thus, long-term experiments
with more than 10 years of continuous treatments are often used to
infer on the effects of land use change, tillage and fertilization practices
on soil organic matter dynamics, so their cumulative effects could be
distinguished and properly evaluated (Angers and Eriksen-Hamel,
2008; Kopittke et al., 2017; Luo et al., 2010). Nonetheless, most of SOC
losses following the conversion of natural soils to agriculture were re-
ported to occur within the first 10 years after land use change, espe-
cially in tropical soils (Deng et al., 2016; Wei et al., 2014). Moreover,
West and Post (2002) showed that changes on TOC stocks can be ex-
pected to peak just between 5 to 10 years after the adoption of CA
practices, such as NT and crop rotation. In this sense, short-term as-
sessments, such as our study (5 years), may be useful to infer on the
potential of CA practices to mitigate TOC and TN losses following the
conversion of natural grasslands to agriculture under contrasting tillage
and fertilization practices.
As expected, the disruption of the grassland soil followed by just 5
years of continuous CT decreased TOC in both upper (0−10 cm) and
deeper soil layers (30−60 cm). Nonetheless, the processes concurring
for TOC losses were different according to soil depth. Mixing of C-rich
surface layers with C-depleted subsurface layers by tillage operations
and increased TOC mineralization rates likely concurred for depleting
stocks at the surface layers of CT soils (Bayer et al., 2006; Grave et al.,
2015; Nicoloso et al., 2016). In contrast, redistribution of aboveground
C inputs with tillage operations sustained TOC stocks at the 10−30 cm
layers of the same treatment (de Oliveira Ferreira et al., 2013;
Franzluebbers, 2002; Nicoloso et al., 2018). In turn, TOC losses ob-
served at the 30−60 cm soil layers of both CT and NT soils were related
with decreased root C inputs from maize and black oats in comparison
with the perennial grassland vegetation with deeper root system
(Dietzel et al., 2017).
Several studies already discussed the importance of root C inputs for
TOC at depth under different cropping systems (Adkins et al., 2016;
Baker et al., 2007; Dietzel et al., 2017; Mcgowan et al., 2019). In our
study, maize root biomass measured in the 0−10 cm soil layer ac-
counted for about 90 % of the root biomass found within the 0−30 cm
soil layer, on the average of CT and NT plots (data not shown). Thus, in
addition to the shallower root system of maize and black oats as com-
pared with the grassland vegetation, subsoil acidity may have further
impaired root growth and C inputs to deeper soils layers, regardless of
tillage system, as often observed in weathered tropical soils (Dalla Nora
et al., 2017b; Dalla Nora and Amado, 2013).
Total OC stocks decreased by 9 % in the 0−60 cm soil layer of CT
soils in comparison with the grassland soil. Losses of TOC by centimeter
of soil were relatively greater at the upper soil layers (Luo et al., 2010),
yet 65 % of the TOC losses were observed at the 30−60 cm soil layers.
In contrast, NT increased TOC at the soil surface (0−5 cm), with a
significant accrual of 2.9 Mg TOC ha−1
in the cumulative 0−30 cm soil
layer, on the average of N sources. However, TOC losses at depth offset
gains at the surface layers with NT, resulting in no difference on TOC
stocks as compared with the grassland soil. Our results reinforce the
importance of deep soil sampling (> 30 cm) to investigate TOC dy-
namics due to land use change and soil tillage practices (Baker et al.,
Table 4
Sand-free water-stable aggregates in the 0-0.05 m layer of a Nitisol from
Southern Brazil as affected by five years of continuous conventional tillage (CT)
and no-till (NT) amended with urea (UR), composted swine slurry (CS), besides
a control treatment without N (CTR).
Tillage N source Sand-free water-stable aggregates (μm) Sample
Recovery
< 53 53−250 250−2.000 > 2.000
————————————————— g kg−1
soil —————————————————
– % –
CT CTR 455 933 3566 1302 98.6
UR 556 971 3843 1733 99.5
CS 444 753 3258 1922 97.2
NT CTR 245 307 2267 3078 96.6
UR 181 234 1956 3201 96.2
CS 183 307 1361 3295 96.5
Mean CT 485nsC1
886aC 3555aA 1652bB 98.5ns
NT 203C 283bC 1861bB 3191aA 96.4
Mean CTR 350ns 620ns 2917ns 2190ns 97.6ns
UR 368 603 2897 2466 97.9
CS 313 530 2301 2609 96.9
1
Means followed by different lowercase letters in the same column and
uppercase letters in the same line are significantly different by the Tukey-
Kramer test (p < 0.05).
Fig. 4. Soil C and N contents within sand-free water-stable aggregates (WSA)
fractions (< 53, 53-250, 250-2.000 and > 2.000 μm) in the 0-5 cm layer of a
Nitisol from Southern Brazil as affected by five years of continuous conven-
tional tillage (CT), no-till (NT) amended with contrasting N sources. CTR:
control without fertilization; UR: urea; CS: composted pig slurry; Means fol-
lowed by different lowercase letters within the same WSA fraction for the
comparison soil tillage systems or N sources and uppercase letters within the
same soil tillage systems or N sources for the comparison of WSA fractions are
significantly different by the Tukey-Kramer test (p < 0.05). Vertical error bars
indicate the means’ standard error (n=4).
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
8
9. 2007; Lal, 2009; Luo et al., 2010). Moreover, amelioration of subsoil
acidity seems to be crucial to improve root growth and sustain TOC
stocks in deeper soil layers of tropical soils (Dalla Nora et al., 2017b; de
Oliveira Ferreira et al., 2018a). Nonetheless, early adoption of NT mi-
tigated 82 % of TOC losses as compared with the soils under CT in the
measured soil profile (0−60 cm).
Bayer et al. (2006) reported that minimum C inputs to sustain TOC
in the 0–17,5 cm layer of an Acrisol from Southern Brazil under NT and
CT are 3.9 and 8.8 Mg C ha−1
yr−1
, respectively. Mafra et al. (2014)
found similar requirements for the 0−20 cm soil layer of an Oxisol
from Southern Brazil under NT, amended with either mineral fertilizers
or pig slurry (4.1 Mg C ha−1
yr−1
). Aboveground C inputs in our study
ranged from 6.2–8.9 Mg C ha−1
yr−1
according to N sources. Thus,
losses and accumulation of TOC were expected to occur under CT and
NT, respectively, as observed in the 0−30 cm soil layer. Since C hu-
mification were likely similar in CT and NT soils, our results suggest
that adoption of NT decreased TOC mineralization at the surface layers
after disruption of the grassland soil thus allowing TOC accrual (Bayer
et al., 2006; Grave et al., 2015; Huggins et al., 2007; Nicoloso et al.,
2016; Pes et al., 2011). Moreover, our results confirm that NT can not
only recover TOC up to levels of soils under natural vegetation (de
Oliveira Ferreira et al., 2018b, 2016), but given sufficient C inputs NT
soils can build up TOC exceeding natural levels towards the saturation
of topsoil layers (Briedis et al., 2018; Nicoloso et al., 2020, 2018).
Nonetheless, significant losses of TN (21 % or original stocks or 2.6
Mg TN ha−1
) were observed throughout the soil profile under CT, with
the 0−30 and 30−60 cm soil layers accounting for 52 and 48 % of the
observed losses, respectively. The use of NT mitigated TN losses by 34
% as compared with CT (0−60 cm), mostly by decreasing losses at
surface soil layers (0−10 cm). Yet, significant TN losses were observed
for both 0−30 and 30−60 cm soil layers of NT (−0.6 and −2.6 Mg TN
ha−1
, respectively) in comparison with the grassland soil. Nitrogen
sources had no effect on TOC and TN stocks as well on the soil C/N ratio
throughout the soil profile, regardless of the increased C inputs with the
use of organic fertilizers.
Soil C/N ratio increased from 10.6 ± 0.4 (mean ± 95 %C.I.) in the
grassland soil to 12.2 ± 0.2 on the average of CT and NT as measured in
the 0−60 cm soil layer (Fig. S1). These results indicate decoupling of
C/N cycles due to conversion of grassland to agriculture (Asner et al.,
1997). Decoupling was likely promoted by changes on C and N inputs
quality and quantity to the soil, soil disruption increasing TOC and
moreover TN mineralization rates, and possibly increased N scavenge
by agricultural crops in comparison with the grassland vegetation
(Banegas et al., 2019; Hobley et al., 2018). Nonetheless, after 5 years of
continuous CT and NT, TOC had strong correlations with and TN stocks
in both 0−5 and 0−30 cm soil layers (r > 0.94, p < 0.01, Table 5).
These results suggest that TOC/TN recoupled at higher C/N ratios levels
after the stabilization of agricultural soils under continuous tillage and
fertilization practices (Asner et al., 1997).
4.2. Particulate and mineral associated C and N fractions
Previous studies reported losses of POC and PN fractions when
natural vegetation was replaced with agroecosystems in response to
increasing soil disturbance (Figueiredo et al., 2013; Six et al., 1998;
Tivet et al., 2013a). We observed significant losses of POC in the 0−5
cm and PN in the 0−5 and 5−10 cm layers of CT soils as compared
with the grassland. In contrast, NT had higher POC and PN in the 0−5
and 5−10 cm layers than both CT and grassland soils (Amado et al.,
2006; Bayer et al., 2004; Figueiredo et al., 2013; Hok et al., 2015; Oorts
et al., 2007; Salvo et al., 2010). Other studies already suggested that NT
under increased C inputs as promoted by crop rotation and fertilization
could recover POC stocks up to the levels of soils under natural vege-
tation (Amado et al., 2006; Blanco-Moure et al., 2013; Sainju et al.,
2008). Thus, increased aboveground carbon inputs from maize and
black oats and possibly by organic fertilizers (as will be discussed later
using the measured soil C and N quality indices) favored POC and PN
accumulation in the surface of NT soils surpassing the levels of the
grassland soil. Interestingly, both tillage systems had increased POC
and PN in the 10−20 cm layer as promoted by residue incorporation
following the conversion of the grassland to agriculture in 2012 (NT)
and continuous soil disruption in CT treatment (Bayer et al., 2004;
Campos et al., 2011; Franzluebbers, 2002). Thus, both CT and NT soils
had significant increases on POC and PN in the 0−30 cm soil layer as
compared with the grassland soil.
Differences among tillage systems were also observed for the MAOC
and MAN fractions yet only in the 0−5 cm soil layer. As compared with
the grassland, CT soils lost MAOC and MAN in the 0−5 and 5−10 cm
layers thus resulting in significant losses in the cumulative 0−30 cm
layer. No-till soils also lost MAN in the same 0−5, 5−10 and 0−30 cm
layers yet MAOC losses were only observed in the 5−10 cm layer, re-
sulting in no significant differences in the cumulative 0−30 cm soil
layer as compared with the grassland. Changes of MAOC were more
discrete than those observed for POC (up to 2.1 and 7.5 Mg C ha−1
,
respectively, in the 0−30 cm soil layer) confirming the lower sensi-
tivity of this fraction as reported elsewhere (Campos et al., 2011;
Figueiredo et al., 2013; Oorts et al., 2007; Reis et al., 2014; Zanatta
et al., 2019). Soil C and N associated with silt + clay fractions are
strongly protected from microbial decomposition due to organo-mi-
neral adsorption thus increasing stabilization of MAOC and MAN
fractions, especially in oxidic tropical soils with (Briedis et al., 2018;
Razafimbelo et al., 2008). Nonetheless, losses of the mineral associated
fractions were expected in response land use change while mitigated by
decreased soil disturbance and increased C inputs as reported in pre-
vious studies (Reis et al., 2014; Tivet et al., 2013a).
4.3. Soil C and N quality indices
Nitrogen sources had no significant effect on TOC and TN stocks
throughout the soil profile probably because the changes observed
during our short-term study (5 years) were relatively small in com-
parison with high TOC and TN background levels of the grassland soil
and natural soil variability (Blair et al., 1995). Nonetheless, the use of
CS increased the SR of both TOC and TN as well of both POC and PN
pools. Stratification of TOC (i.e. the ratio between TOC contents in the
soil surface and a underlying soil layer) and other soil organic matter
pools was proposed as a sensitive indicator of soil quality and C dy-
namics in both temperate and tropical soils (de Oliveira Ferreira et al.,
2013; Franzluebbers, 2002; Zanatta et al., 2019). Higher SR were re-
ported in soils under natural ecosystems as well in agricultural soils
under minimum disturbance and increased C inputs (Franzluebbers,
2002). De Oliveira Ferreira et al. (2013b) suggested that TOCSR values
of 1.5–1.7 indicates high quality tropical agroecosystems managed
under conservation agriculture practices.
The SR measured in the grassland soil for both TOC and TN as well
for the particulate C and N fractions fell within this range (1.56–1.70),
indicating a high quality soil. The SR measured for the same total and
particulate fractions ranged from 1.82 to 1.99 in NT soils and 1.27–1.36
under CT, thus indicating that NT favored soil quality while CT pro-
moted soil degradation. Soils amended with CS had SR increased to
1.81–2.19 for the same total and particulate C and N fractions, on the
average of tillage systems. No such effect was noticed for the others N
sources. Our results suggests that continued application of CS may
promote on the long-term significant accrual of TOC and TN stocks
while improving soil quality as already observed in previous assess-
ments (Mafra et al., 2014; Nicoloso et al., 2018; Powlson et al., 2012;
Xia et al., 2017).
In contrast to previous studies (Alvarez et al., 2014; de Oliveira
Ferreira et al., 2013), we did not find correlations for TOCSR and TNSR
with TOC and TN stocks in the 0−30 cm soil layer (Table 5). None-
theless, correlations between these variables were significant for the
0−5 cm soil layer (r > 0.86, p < 0.01), since most of the changes on
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
9
10. TOC and TN stocks due to soil tillage systems were observed in this
layer. In turn, POCSR and PNSR were correlated with TOC and TN in
the 0−5 cm soil layer (r > 0.71, p < 0.01) while only POCSR was
correlated with TOC in the 0−30 cm soil layer (r = 0.33, p < 0.05).
These results indicate that TOC and TN accrual in the surface layers of
NT soils depended on the stabilization of fresh added C from crop re-
sidues and organic fertilizers as POC and PN, which also augmented the
lability of TOC and TN stocks (Zanatta et al., 2019).
In contrast to SR, the CPI or NPI are calculated as a ratio of TOC or
TN stocks in a given treatment to the stocks of a natural soil used as a
reference (Blair et al., 1995). Thus CPI or NPI < 1 indicate losses of
TOC or TN in comparison with the reference soil while CPI or NPI > 1
indicate TOC or TN accumulation (Campos et al., 2011; de Oliveira
Ferreira et al., 2013; Zanatta et al., 2019). Soils under CT had CPI and
NPI of 0.91 and 0.79, respectively. No-till soils did not differ from the
grassland as indicated by the CPI (1.03) but had lower NPI (0.91).
Nonetheless, NT soils had consistently better CPI and NPI than CT soils
(Campos et al., 2011; De Bona et al., 2008; de Oliveira Ferreira et al.,
2013; Zanatta et al., 2019).
The CLI and NLI represent the ratio of the respective POC/MAOC
and PN/MAN fractions in a given treatment in reference to the ratio of
the same fractions in the reference soil (e.g. grassland soil) (Blair et al.,
1995). Both CLI and NLI were sensitive to discriminate tillage systems
but not for the N sources tested in our study, with NT presenting greater
CLI and NLI than CT soils (Campos et al., 2011; Zanatta et al., 2019).
Significant correlations were observed for CLI with TOC and TN in the
0−5 cm soil layer (r > 0.53, p < 0.01, Table 5) and NLI with TOC and
TN in both 0−5 (r > 0.59, p < 0.01) and 0−30 cm soil layers
(r > 0.33, p < 0.05). Nonetheless, both CT and NT soils had increased
CLI and NLI as compared with the grassland soil although through
different processes. The increase in CLI and NLI in NT soils was mostly
due to increase of POC and PN fractions although some MAOC and
MAN losses were observed. In contrast, losses of MAOC and MAN
fractions were more pronounced in CT soils thus also resulting in in-
creased CLI and NLI. The same processes were observed in Acrisol from
southern Brazil where the association of NT with N fertilization and
legume cover crops increased labile C stocks (analogous to particulate C
and N fractions) and consequently its CLI as compared with a natural
grassland soil (Zanatta et al., 2019). The same study reported decreased
labile C stocks and CLI in CT soils without N fertilization and legumes.
De Bona et al. (2008) also suggested that CT and irrigation might de-
crease CLI in comparison with grassland soils by decreasing labile C
pools as compared with the grassland although no differences were
reported for non-labile C pools.
The CMI and NMI are the product of the respectives C and N pools
and lability indices. The CMI was proposed to infer on soil quality and
the rate of change of TOC stocks in managed soils in comparison with
more stable ecosystems, such as natural grasslands or forests soils (Blair
et al., 1995). The CMI was shown to be sensitive to soil tillage, fertili-
zation and other agricultural practices presenting good correlations
with crop biomass production and grain yields, as well with soil
available N, microbial biomass, respiration, aggregate stability, labile C
and TOC stocks (Chatterjee et al., 2018; Vieira et al., 2007; Zanatta
et al., 2019). Nonetheless, both CMI and NMI did not detect differences
among the N sources tested in our study. No-till soils had higher CMI
(1.43) and NMI (1.40) than CT and the grassland soils. In contrast, CT
soils had CMI (1.02) similar to the grassland whereas increased CLI
compensated decreased CPI. Although soils under CT had increased NLI
in comparison with the grassland soil, reduction on the NPI due to TNN
losses resulted in decreased NMI (0.91) in CT soils. Although there is no
ideal range for CMI, positive differences between treatments and over
time indicate improvement of soil quality, while decreasing CMI sug-
gest soil degradation (Blair et al., 1995).
The CMI was considered a good indicator of soil and management
quality by showing good correlations with several soil biological, che-
mical and physical parameters, as reported in previous studies
(Chatterjee et al., 2018; De Bona et al., 2008; Vieira et al., 2007;
Zanatta et al., 2019). We found that both CMI and NMI had significant
correlations with TOC and TN in the 0−5 (r > 0.69, p < 0.01) and
0−30 cm soil layers (r > 0.51, p < 0.01) (Table 5). Thus, both CMI and
NMI consisted on useful indicators to assess the effects of tillage systems
and N sources on soil C and N pools even in short-term studies (5 years)
where measurements of TOC and TN stocks and their respective parti-
culate and mineral associated fractions usually fail to detected differ-
ences among treatments.
Table 5
Pearson's correlation matrix between TOC and TN stocks, soil C and N quality indices and soil C and N within large macroaggregates.
TOC TOC TN TN TOCSR POCSR CPI CLI CMI TNSR PNSR NPI NLI NMI LM LM LM
0−5 0−30 0−5 0−30 Mass C N
TOC 0−5 1.00 0.67§
0.99§
0.64§
0.90§
0.79§
0.73§
0.58§
0.73§
0.87§
0.77§
0.70§
0.63§
0.74§
0.65§
0.78§
0.79§
TOC 0−30 1.00 0.67§
0.94§
0.30 0.33†
0.98§
0.27 0.58§
0.24 0.30 0.94§
0.38†
0.62§
0.43†
0.41†
0.41†
TN 0−5 1.00 0.68§
0.89§
0.72§
0.72§
0.53§
0.69§
0.86§
0.71§
0.73§
0.59§
0.73§
0.65§
0.78§
0.78§
TN 0−30 1.00 0.31 0.24 0.91§
0.22 0.51§
0.23 0.23 0.98§
0.33†
0.61§
0.39 0.35 0.36
TOCSR 1.00 0.83§
0.38†
0.58§
0.60§
0.98§
0.83§
0.37†
0.58§
0.59§
0.56§
0.78§
0.78§
POCSR 1.00 0.40†
0.55§
0.58§
0.80§
0.98§
0.31†
0.55§
0.53§
0.43†
0.68§
0.67§
CPI 1.00 0.37†
0.67§
0.31 0.38†
0.95§
0.47§
0.70§
0.53§
0.49†
0.50†
CLI 1.00 0.93§
0.54§
0.55§
0.31 0.98§
0.89§
0.20 0.36 0.37
CMI 1.00 0.54§
0.58§
0.61§
0.95§
0.98§
0.36 0.46†
0.47†
TNSR 1.00 0.79§
0.28 0.54§
0.52§
0.56§
0.78§
0.78§
PNSR 1.00 0.30 0.56§
0.54§
0.41†
0.66§
0.65§
NPI 1.00 0.41§
0.68§
0.48†
0.43†
0.44†
NLI 1.00 0.94§
0.24 0.35†
0.36†
NMI 1.00 0.35 0.41†
0.42†
LM Mass 1.00 0.88§
0.87§
LM C 1.00 0.99§
LM N 1.00
TOC: total organic carbon stocks in the 0−5 and 0−30 cm soil layers; TN: total nitrogen stocks in the 0−5 and 0−30 cm soil layers; TOCSR: TOC stratification ratio;
POC: particulate organic C stratification ratio; CPI: C pool index; CLI: C lability index; CMI: C management index; TNSR: TN stratification ratio; PNSR: particulate N
stratification ratio; NPI: N pool index; NLI: N lability index; NMI: N management index; LM Mass: mass of large macroaggregates (> 2.000 μm); LM C: soil C within
LM; LM N: soil N within LM; n = 40, except for LM that n = 24
†
Significant p < 0.05.
§
Significant p < 0.01.
C.R. Wuaden, et al. Soil & Tillage Research 204 (2020) 104728
10
11. 4.4. Soil C and N within water-stable aggregates
Addition of fresh crop and organic residues to the soil was shown to
induce the formation of macroaggregates (> 250 μm) at similar rates in
CT and NT soils (Jastrow, 1996; Six et al., 1999). Six et al. (2000)
suggested that protection of POC within macroaggregates and its sta-
bilization within microaggregates is the mechanism regulating SOC
accrual in NT soils. Nonetheless, the authors also proposed that dis-
ruption of macroaggregates by tillage operations increases POC de-
composition due to reduced physical protection with increased mac-
roaggregates turnover, thus promoting SOC losses in CT soils. We found
similar masses of macroaggregates (> 250 μm) in CT and NT soils with
respective 520 and 505 g kg−1
soil. Nonetheless, the use of NT nearly
doubled the mass of large macroaggregates (> 2000 μm) in comparison
with CT soils (319 and 165 g kg−1
soil, respectively) as already re-
ported for both temperate and tropical soils (Chung et al., 2008;
Conceição et al., 2013; Fabrizzi et al., 2009; Mikha and Rice, 2004;
Nicoloso et al., 2018; Tivet et al., 2013b). Other aggregate size fractions
were proportionally diluted in NT soils as compared with CT.
Previous studies reported that the mass of large macroaggregates
was closely correlated with TOC accrual under NT in both temperate
and tropical soils (de Oliveira Ferreira et al., 2018b; Fabrizzi et al.,
2009; Tivet et al., 2013b). We found similar contents of C and N as-
sociated with the silt + clay, microaggregates and macroaggregates
fractions on the comparison of soil tillage systems or N sources. In
contrast, NT soils had three times more C and N within the large
macroaggregates fraction than CT soils. Thus, we found evidences
supporting that POC and PN protection within large macroaggregates
was the mechanism regulating TOC accrual and preventing TN losses
under NT. Frequent disruption of large macroaggregates with CT in-
creased mineralization of both particulate and mineral associated
fractions, thus decreasing TOC and TN at the soil surface layer (de
Oliveira Ferreira et al., 2018b; Fabrizzi et al., 2009; Nicoloso et al.,
2018; Tivet et al., 2013b).
Although N sources did not affect the mass of any of WSA fractions,
the use of CS augmented C and N contents within large macro-
aggregates by respective 35 and 40 % in comparison with UR and by 72
and 86 %, respectively, if compared with the CTR treatment.
Coincidently, continuous application (> 30 years) of increasing rates of
cattle manure (up to 180 Mg ha−1
yr−1
) did not affect the proportion of
large macroaggregates in the 0−15 cm layer of a Mollisol from Canada
under CT (Gulde et al., 2008). Nonetheless, the same study reported
increased POC contents within large macroaggregates and macro-
aggregates fractions (LM > M) and C within the large macroaggregates,
macroaggregates and microaggregates fractions (LM > M > m), which
were linearly correlated with TOC as measured in the bulk soil at the
same soil depth. Nicoloso et al. (2018) also showed that long-term
application of cattle manure (10 years) followed by composted organic
waste (17 years) increased C within all WSA fractions in a Mollisol from
central Kansas under NT up to the levels of nearby native prairie (0−5
cm). The short duration of our study (5 years) associated with higher
aggregate stability of tropical soils as compared with temperate soils
(Briedis et al., 2018; Fabrizzi et al., 2009) probably prevented the re-
distribution of C and N derived from crop residues and CS from the
large macroaggregates to other WSA fractions (Nicoloso et al., 2018; Six
et al., 2000). Soils under long-term manure application often present
evidence of C saturation as reported in previous studies in both CT and
NT soils when C levels within WSA approaches the levels of soils under
grassland or forests (de Oliveira Ferreira et al., 2018b; Gulde et al.,
2008; Nicoloso et al., 2018). Nonetheless, the higher saturation deficit
of tropical soils and the formation of large macroaggregates protecting
POC and PN allowed for increased TOC stocks in the surface layer of NT
soils as compared with the grassland (de Oliveira Ferreira et al., 2016;
Reis et al., 2014).
The mass of large macroaggregates and the C and N contents as-
sociated with this WSA fraction had significant correlations with TOC
and TN stocks in the 0−5 cm soil layer (r > 0.65, p < 0.01) and with
TOC stocks in the 0−30 cm soil layer (r > 0.41, p < 0.05) (Table 5).
The mass of large macroaggregates was also correlated with other soil C
quality indices, such as the TOCSR, POCSR, CPI (r > 0.43, p < 0.05)
and the corresponding N indices (r > 0.41, p < 0.05). The C and N
contents within large macroaggregates were correlated with the same
indices (r > 0.43, p < 0.05) as well with CMI (r > 0.46, p < 0.05), NLI
(r > 0.35, p < 0.05) and NMI (r > 0.41, p < 0.05). Soil aggregate sta-
bility and the proportion of macroaggregates were already correlated
with the stratification ratio of both total and particulate C fractions, as
well with the CMI as reported in previous studies (Vieira et al., 2007;
Zanatta et al., 2019). These results reinforce the importance of C and N
protection within large macroaggregates to promote soil quality and
TOC and TN storage at the soil surface layers (de Oliveira Ferreira et al.,
2018b; Fabrizzi et al., 2009; Nicoloso et al., 2018; Tivet et al., 2013b).
5. Conclusions
The conversion of a Nitisol from Southern Brazil under natural
grassland to agriculture with CT decreased TOC and TN by respective 9
and 21 % (0−60 cm) in just 5 years after land use change. Early
adoption of NT mitigated TOC and TN losses by 82 and 34 % as com-
pared with soil under continuous CT. No tillage also increased TOC and
prevented TN losses at topsoil layers (0−30 cm) by increasing the mass
of large macroaggregates and promoting accumulation of POC and PN.
Both NT and the use of CS increased the stratification ratio of total and
particulate C and N stocks and augmented C and N protection within
large macroaggregates. Soil quality indices, the mass of large macro-
aggregates and the C and N contents associated with this WSA fraction
were indicative TOC and TN accrual in the soil surface (0−30 cm) even
in short-term assessments (5 years) such as the present study.
Nonetheless, both CT and NT soils had significant losses of TOC and
TN in deeper soil layers (30−60 cm), respectively aggravating losses
and offsetting gains at surface layers. Substitution of natural grassland
vegetation by agricultural crops with shallower root system impaired
root C inputs to deeper soil layers thus promoting TOC and TN losses.
Our results suggest that amelioration of subsoil acidity and the use of
crops with deep root systems are necessary to sustain TOC stocks in
deeper layers of tropical soils following its conversion to agriculture.
Moreover, our study reinforce the importance of deep soil sampling
(> 30 cm) to investigate TOC dynamics due to land use change and soil
tillage practices even in short periods.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors thank the Brazilian Agricultural Research Corporation
(Embrapa) under project no. 03.17.00.032.00.00 and the Brazilian
National Council for Scientific and Technological Development (CNPq)
under grants nos. 401196/2016-0 and 302189/2018-1 for funding this
research.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at doi:10.1016/j.still.2020.104728.
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