Soil physical and biological quality in a peasant

agroforestry system in Southern Brazil

Aghata C. R. Charnobay (  aghatacharnobay@gmail.com )
Londrina State University
Pedro H. Weirich Neto
Ponta Grossa State University
André C. Auler
Federal University of Paraná
Jaime A. Gomes
Ponta Grossa State University
Natali M. de Souza
Ponta Grossa State University
Daiane Hyeda
Ponta Grossa State University
Carolina W. Galvão
Ponta Grossa State University
Aline L. Gomes
Ponta Grossa State University
Carmen L. R. F. Lima
Ponta Grossa State University

Research Article

Keywords: Soil health, Soil biology, Agroecology, Family farming, VESS

Posted Date: February 21st, 2023

DOI: https://doi.org/10.21203/rs.3.rs-2239028/v2

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Agroforestry systems bring together social, economic, and environmental services and can be an
alternative for promoting development and sustainability in peasant agriculture. This study aimed to
evaluate the soil quality in a peasant agroecological agroforestry intercropped with vegetables (South
Brazil). Treatments included an agroforestry row (AFS-R) managed under no-tillage and cultivated with
trees for fruit and wood production; an agroforestry inter-row (AFS-I) managed with conventional tillage
and cultivated with vegetables; and an area managed with conventional tillage (CT). Soil physical
assessments comprised soil bulk density, total porosity, macroporosity, microporosity, bioporosity, mean
weight diameter of aggregates, field capacity, aeration capacity, soil saturated hydraulic conductivity, and
the visual evaluation of soil structure. Microbiological indicators included soil microbial biomass, basal
respiration, microbial quotient, metabolic quotient, soil organic carbon, and the most probable number of
nitrogen-fixing, ammonifying, and nitrite-oxidizing bacteria. The results demonstrated that AFS-R
management improved several soil physical and microbiological attributes, highlighting the importance
of plant diversity, organic residue inputs, and no-tillage for soil quality. On the other hand, the tillage
practice contributed to structural and microbiological degradation in CT, and more intensively in AFS-I,
indicating that more conservationist soil management, like minimum or no-tillage, might be studied as an
alternative for the cultivation of vegetable crops in agroforestry systems.

Introduction
The pressure to increase land productivity is leading to land use intensification by agriculture, with
increased use of irrigation, fertilizers, pesticides, and mechanization (Foley et al., 2011), as well as
inadequate crop rotation, overgrazing or removal of crop residues (Karlen and Rice, 2015). These
practices may contribute to decreasing soil quality (SQ). Soil structure is a component of SQ and
regulates processes of water infiltration and retention, gas exchange, nutrient dynamics, and
susceptibility to erosion (Rabot et al., 2018), as well as biological attributes such as microbial biomass
(Silva et al., 2014; Merlo et al., 2022) and activity (Cui and Holden, 2015).

Agroforestry systems (AFS) are ancient and traditional production systems that are being brought back to
current land use and research scenarios to take advantage of their sustainability attributes and benefits
(Nair, 2014). AFS consists of the simultaneous cultivation of trees, agricultural species, and sometimes
animals in the same cropping area, arranged in interactive combinations to generate a variety of products
and goods, as well as environmental and ecological services (Nair et al., 2017). Studies have shown that
AFS contribute to the conservation of biodiversity (Udawatta et al., 2019), air and water quality (Jose,
2009), carbon sequestration (Eddy and Yang, 2022) and the reduction of native forests deforestation
(Sharma et al., 2016).

Soil physical quality increases in ASF, as a consequence of the action of roots and soil organisms, and
organic matter accumulation, which helps decreasing soil erosion processes and improving water quality
and quantity (Silva et al., 2011; Guimarães et al., 2014; Cherubin et al., 2019).The constant addition of

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organic matter from tree pruning and the action of the different sizes and shapes of roots also stimulates
nutrient availability and cycling and soil organisms in the ASF, thus improving soil fertility (Froufe et al.,
2020; Matos et al., 2022) and biology (Cezar et al., 2015; Beule et al., 2022).

The adoption of AFS may also contribute to economic and social aspects, being promoted as a
sustainable production alternative for smallholder farming in Brazil (Bezerra et al., 2019; Shennan‐Farpón
et al., 2022). The great diversity of food production in AFS, which comes from annual and vegetable
crops, forestry and fruit species harvested at different times of the year, increases smallholder farmer’s
income and optimizes family labour. In South Brazil, experiences with agroforestry systems inter-rowed
with annual or vegetable crops are carried out in many smallholder farms (Stöcker et al., 2020; Valani et
al., 2020).

Soil management of the AFS inter-rows, where vegetables are annually grown, can vary from the
management of the rows, where trees are cropped. Vegetable production relies highly on topsoil turning
and mixing to prepare the beds for crop growth, especially in organic and agroecological systems (Costa
et al., 2018). The impacts of conventional tillage on soil physical attributes in grain and vegetable
production systems are well known (Moraes et al., 2016; Norris and Congreves, 2018; Nunes et al., 2020),
but there is little information regarding the effects of this kind of practice on the structural and
microbiological soil attributes in agroforestry systems. This study aimed to evaluate the soil structural
and biological quality of an agroecological agroforestry in South Brazil.

Material And Methods

2. 1 Area and treatments

The study was carried out in an agroecological smallholder farm at the Contestado Settlement
(25º37’09” S and 49º42’07” W, 910 m above sea level), in the city of Lapa – Paraná State, Brazil. The
climate of the region is classified as Cfb (Köppen) (humid subtropical), with annual average rainfall
ranging from 1400 to 1600 mm and average annual temperature from 17 to 18°C (Caviglione et al.,
2000). The soil is an Inceptisol (Soil Survey Staff, 2014) or Cambissolo Háplico Tb Distrófico (Brazilian
Soil Classification) (Santos et al., 2018).

At the farm, soil samples were taken in three areas with different soil management: agroforestry
row (AFS-R), agroforestry inter-row (AFS-IR), and area managed with conventional tillage (CT).
Granulometric and chemical characteristics of the areas are described in Table 1.

Table 1. Granulometry and chemical characterization of the soil (0-0.2 m) under the

areas: agroforestry-row (AFS-R), agroforestry inter-row (AFS-I), and conventional tillage

(CT), located in Lapa – PR, Brazil

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 Soil attributes AFS-R AFS-I CT

Clay (g kg-1) 268.0 273.3 356.8

Particle density (g cm-3) 2.4 2.3 2.3

pHa 5.3 5.4 5.1

Ca2+ (cmolc dm-3)b 4.4 4.5 5.9

Mg2+ (cmolc dm-3) 2.4 2.6 2.8

K+ (cmolc dm-3) 0.4 0.5 0.1

Al3+ (cmolc dm-3) 0.0 0.0 0.1

H + Al (cmolc dm-3) 5.36 5.48 7.28

P (mg dm-3) 10.6 7.5 11.8

a
measured in 0.01 mol L-1 CaCl2, bExchangeable calcium (Ca2+), exchangeable magnesium

(Mg2+), exchangeable potassium (K+), aluminium (Al3+), potential acidity (H + Al), phosphorus

(P)

The agroforestry system was established in 2015 in 0.5 ha on a dairy cattle pasture area. Soil was tilled
with plowing and harrowing, lime was applied, and then oat (Avena sativa L.) and vetch (Vicia sativa L.)
cover crops were sown. Twelve tree rows were implemented every 6 meters in an East-West direction. In
the AFS-R an additional chiseling operation was performed before transplanting, being the soil tilled only
at that time. Species for feeding (Acca sellowiana ((O.Berg) Burret), Actinidia deliciosa (A.Chev)), Citrus
limon (L.), Citrus reticulata (L.), Citrus sinensis (L.), Eriobotrya japonica ((Thunb.) Lindl.), Eugenia
uniflora (L.), Ilex paraguariensis (A.St.-Hil.), Manihot esculenta (Crantz), Musa spp., Passiflora
edulis (Sims), Prunus persica, Prunus persica var. nucipersica ((Suckow) C. K. Schneid.0, Psidium
cattleianum (Sabine), Schinus terebinthifolius), and for wood (Anadenanthera colubrina ((Vell.) Brenan),
Cedrela fissilis (Vell), Eucalyptus sp., Toona ciliate (M.Roem.)) and biomass production (Eucalyptus sp.,
Morus sp., Musa spp., Sida sp.) are cropped in the rows.

The agroforestry inter-rows (AFS-I) are cultivated with vegetable crops (Allium cepa (L.), Beta vulgaris L.,
Brassica oleracea L., B. oleracea var. botrytis L., B. oleracea var. italica Plenck, Daucus carota (L.),
Dioscorea sp., Eruca sativa (Mill.), Lactuca sativa (L.), Manihot esculenta (Crantz), Solanum
lycopersicum (L.)) and beds are prepared annually with a rotary tiller. AFS-I received turkey litter (4.8 Mg
ha-1 year-1) as organic fertilizer and was supplemented with mineral potassium sulphate (0.72 Mg ha-
1
year-1). Turkey litter in AFS-I was incorporated on soil and potassium sulphate were broadcasted to the
soil surface and covered with crop residues mowed from the AFS-R tress.

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The area under CT covered approximately 0.3 ha and was in fallow before cultivation. Soil was tilled with
plowing and leveling harrow 7 months before the experimental evaluations. The crop present at the time
of sampling was a landrace corn (Zea mays). After the emergence of corn, fertilization occurred in total
area with turkey litter (1.2 Mg ha-1 year-1).

2.2 Soil sampling and analysis

Each management system was split into six plots and one point per plot was sampled. The
undisturbed samples were collected at the 0–0.1, 0.1–0.2 and 0.2–0.4 m soil depths, in volumetric rings
(0.05 × 0.05 m of internal diameter and height) using an Uhland auger. Soil monoliths (0.1 × 0.1 m) for
aggregate stability analysis were collected at the 0-0.1 m and 0-0.2 m. For soil texture and chemical
characterization, five soil disturbed sub-samples were taken at the 0-0.2 m to compose one sample
(Teixeira et al., 2017).

Soil sampling for microbiological analysis was carried out at the 0-0.1 m. Six disturbed samples, formed
through 10 sub-samples, were collected close to the points of previous soil sampling. At the laboratory,
soil biological samples were homogenized, sieved (2 mm) and stored in plastic bags at 4° C until
analysis.

The Visual Evaluation of Soil Structure (VESS) was carried out in the field using the method
proposed by Ball et al. (2007) and improved by Guimarães et al. (2011).

Saturated hydraulic conductivity (Ksf) was measured in the field by the simplified falling-head (SFH)
method (Bagarello et al., 2004). The analysis was carried out at the 0-0.1 m depth, with 12 repetitions due
to Kfs high variability. The water volume used to determine the time of infiltration into the soil was 730
mL and the internal area of the metal ring used was 0.3979 m2 (internal diameter of 0.208 m),
corresponding to a D-value of 0.0214 m (Reynolds and Elrick, 2002). Soil initial volumetric and saturation
moisture content for Ksf analysis were determined in the laboratory. The hydraulic conductivity of the
saturated soil (Kfs) was calculated (Scarabeli et al., 2018).

Measurements of soil bulk density (BD), total porosity (TP), macroporosity (Ma), microporosity (Mi), field
capacity (FC) and aeration capacity (AC) were carried out according to Teixeira et al. (2017) and
bioporosity (Bp) according to Andrade et al. (2016). Bulk density was determined by the core method. TP
was determined by the relationship between BD and particle density. Bp, Mi, and FC were measured
applying the matric heads of -1, -6, and -10 kPa, respectively, in a tension table (model M1-0801,
Eeijkamp®). Ma and AC were estimated based on the differences between Tp in relation to Mi and FC,
respectively. Soil organic carbon (SOC) was determined by the colorimetric method after extraction with
sodium dichromate (Na2Cr2O7 0.667 mol L-1) added in sulfuric acid (5 mol L-1) (van Raij et al., 2001).

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Aggregate stability analysis was carried out by manual fragmentation of the soil monoliths in their
weakness plans, followed by sieving (19 mm mesh). Separation of the soil aggregate classes (8–19; 4–8;
2–4; 1–2; 0.5–1; 0.25–0.5 and 0.053–0.25 mm) was performed by wet sieving (Yoder, 1936) with
adaptations for tropical soils (Castro Filho et al., 1998). The set of aggregates retained in each sieve was
dried at 40 °C, weighted, and the mean weight diameter (MWD) was determined (Castro Filho et al., 1998).
The proportions of the classes of large macroaggregates (L MAgg) - 8–19 mm, medium
macroaggregates (M-MAgg) - 2–8 mm, small macroaggregates (S-MAgg) - 0.25–2 mm, and
microaggregates (MicroAgg) - 0.053–0.25 mm, were also calculated (Ferreira et al., 2018).

Soil microbial biomass of carbon (MBC) was determined by the fumigation–extraction method (Vance et
al., 1987) using an extraction coefficient KC = 0.33 (Brookes et al., 1985). Soil basal respiration (BR) was
estimated according to Alef (1995). The microbial quotient (qMic) was determined by the ratio between
MBC and SOC (Insam and Domsch, 1988). The metabolic quotient (qCO2) was calculated by the ratio
between BR and MBC (Insam and Haselwandter, 1989). The total organic carbon for microbiological
analysis was determined by extraction with potassium dichromate (K2Cr2O7) and subsequent titration
with ammonium ferrous sulfate solution (Walkley and Black, 1934).

The number of nitrogen-fixing bacteria (NFB), ammonifying bacteria (AB), and nitrite-oxidizing bacteria
(NOB) were estimated by the most probable number (MPN) (Hungria and Araujo, 1994).

2.3 Statistical analysis

Data were submitted to analysis of variance. Residue normality and homogeneity of variances were
verified by Shapiro-Wilk and Bartlett tests, respectively. When data transformation was necessary, Box-
Cox optimum potency was used. After presuppositions had been verified, F test was performed and when
significant (P <0.05), the means were compared by the Tukey test at 5% of probability. A principal
component analysis (PCA) was performed to check the relationship among the sampling areas and the
structural and microbiological variables, as well as the relationship among the variables at the 0-0.1 m
soil layer. The VIF (Variance Inflation Factor) test was performed before PCA to exclude from analysis
variables with high collinearity (VIF>20). All statistical analyses were performed using the software R,
version 4.0.2 (R Core Team, 2020).

Results
Soil physical variables and soil organic carbon (SOC) showed significant differences among the
treatments. Soil BD and TP were affected by the treatments only at the 0-0.1 m soil layer (Table 2). AFS-I
increased BD by 7.6 % in relation to AFS-R, which showed the lowest BD value. AFS-R promoted
significant increases in TP, with values 9.8 % higher than AFS-I. CT did not differ from AFS-I and AFS-R for
both variables.

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Table 2. Soil organic carbon (SOC), bulk density (BD), total porosity (Tp), biopores (Bp),

macropores (Ma), micropores (Mi), and mean weight diameter of aggregates (MWD) under

agroforestry-row (AFS-R), agroforestry inter-row (AFS-I), and conventional tillage (CT).

Soil atributes Layer (m) AFS-R AFS-I CT P-value CV (%)

SOC 0-0.10 32.50 31.0 34.1 0.351 11.2

(g kg-1) 0.1-0.2 m 25.83 28.50 30.67 0.07 11.65

0.2-0.4 34.0 a 22.0 c 25.33 b <0.01 20.85

BD 0-0.10 1.05 b 1.13 a 1.07 ab 0.05 4.97

(g cm-3) 0.1-0.2 1.15 1.19 1.12 0.32 7.03

0.2-0.4 1.21 1.24 1.19 0.52 6.80

Tp 0-0.10 0.56 a 0.51 b 0.52 ab <0.05 5.0

(cm3 cm-3) 0.1-0.2 0.50 0.46 0.50 0.15 7.18

0.2-0.4 0.45 0.46 0.50 0.09 7.68

Bp 0-0.10 0.15 a 0.08 b 0.06 b <0.001 8.70

(cm3 cm-3) 0.1-0.2 0.10 a 0.05 b 0.03 c <0.001 20.59

0.2-0.4 0.06 0.06 0.05 0.47 25.94

Ma 0-0.10 0.24 a 0.18 b 0.11 c <0.001 12.3

(cm3 cm-3) 0.1-0.2 0.15 a 0.08 b 0.05 c <0.001 16.7

0.2-0.4 0.09 0.08 0.06 0.06 22.2

Mi 0-0.10 0.32 b 0.31 b 0.43 a <0.001 5.90

(cm3 cm-3) 0.1-0.2 0.35 b 0.39 b 0.45 a <0.001 8.80

0.2-0.4 0.36 b 0.38 b 0.41 a <0.001 6.50

MWD 0-0.10 7.43 ab 7.07 b 7.92 a <0.01 5.35

(mm) 0.1-0.2 8.15 a 6.40 b 7.90 a <0.01 7.41

Means followed by different letters in line are significantly different (Tukey, P ≤0.05).

The TP distribution, represented by Bp, Ma, and Mi, was significantly influenced by the soil management
at the 0-0.1 and 0.1-0.2 m layers, and Mi at the 0.2-0.4 m. AFS-R showed the highest Bp values, increased
by 87.5 % and 150% at the 0-0.1 m, and 94% and 177.1% at the 0.1-0.2 m layers in relation to AFS-I and
CT, respectively. Regarding soil Ma, AFS-R row showed volumes higher than AFS-I and CT at both soil

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layers. CT presented the highest values of Mi compared to AFS-R and AFS-I, which did not differ
statistically from each other. When compared to CT, AFS-I showed higher values of Ma at the 0-0.1 m, and
Ma and Bp at the 0.1-0.2 m layers.

The MWD from AFS-I decreased in relation to CT in all soil layers, and in relation to AFS-R at 0.1-0.2 m
layer (Table 2). For both soil depths MWD from AFS-R and CT did not differ from each other. However, in
AFS-R the results show an increase in the diameter of aggregates of 0.72 mm at 0.1-0. 2 m layer (Table
2). The MWD alterations were a direct effect of the aggregate classes’ distribution (Figure 1). In both soil
layers, CT presented the higher proportion of large macroaggregates (8-19 mm), and AFS-I the lowest
values (P <0.05). At 0.1-0.2 m layer AFS-I had an increase in the proportion of medium macroaggregates
by 21.70 and 18.50 % (P <0.01) in relation to AFS-R and CT, respectively. Small macroaggregates (0.25-2
mm) and microaggregates (<0.25 mm) presented an opposite behavior than large macroaggregates.
AFS-I showed a higher proportion of medium and small macroaggregates and microaggregates
regulating the soil structure, mainly at 0.10-0.20 m, when compared to AFS-R and CT.

The agroforestry row presented an overall VESS Sq of 1.47, significantly better than AFS-I (1.8) and CT
(2.3) (P <0.001) (Figure 2b). When the average score of each individual soil layer was considered, no
differences were found among the treatments for the more superficial soil layer (Figure 2a). However, the
second layer (on average below the 0-0.1 m) preserved the overall Sq order of differences (P <0.001),
demonstrating that AFS-R maintained a better structural quality in depth compared to AFS-I and CT, with
this last one presenting a Sq value of 2.9, which is very close to the limit considered as a good soil
structural quality.

SOC was affected by soil management only at the 0.2-0.4 m soil layer (Table 2). AFS-R showed the
highest value of SOC, near 55 and 34 % higher than AFS-I and CT, respectively. AFS-I presented a lower
SOC than CT at 0.2-0.4 m. It is noteworthy that only for AFS-R, SOC increased 1.04 and 1.32 times at 0.2-
0.4 m soil layer, in relation to 0-0.1 and 0.1-0.2 m layers, respectively (Table 2).

AFS-R presented the highest Kfs value, statistically different and approximately two times greater from
AFS-I, which showed the lowest Ksf. CT did not differ from the other treatments. In relation to FC,
considering the matric head of -10 kPa, CT showed the highest values in relation to AFS-R and AFS-I in all
soil layers (Table 3). However, this variation was more expressive at the 0-0.1 m layer (34 and 39% higher
than AFS-R and AFS-I, respectively) and reduced gradually to 7 and 14% at the 0.2-0.4 m. AFS-R and AFS-I
presented a similar FC, with no significant differences, in all soil layers (Table 3).

Table 3. Field capacity (FC), aeration capacity (AC), and saturated hydraulic conductivity (Kfs)

under agroforestry-row (AFS-R), agroforestry inter-row (AFS-I), and conventional tillage (CT).

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 Soil atributes Layer (m) AFS-R AFS-I CT P-value CV (%)
FC 0-0.10 0.33 b 0.29 b 0.39 a <0.001 8.05

(cm3 cm-3) 0.1-0.2 0.33 b 0.39 b 0.44 a <0.001 9.72

0.2-0.4 0.35 b 0.36 b 0.43 a <0.001 7.13

AC 0-0.10 0.27 a 0.19 b 0.13 c <0.001 6.19

(cm3 cm-3) 0.1-0.2 0.16 a 0.1 b 0.07 b <0.001 14.25

0.2-0.4 0.11 0.10 0.07 0.392 42.80

Kfs (mm h-1) 0-0.10 806.26 a 428.9 b 728.26 ab 0.021 51.44

Means followed by different letters in line are significantly different (Tukey, P ≤0.05).

Regarding AC, the highest differences among treatments were found at 0-0.1 m soil layer (Table 3). In this
layer, AFS-R showed an AC 42.1 and 107.7 % higher than AFS-I and CT, respectively. AC from CT at 0-0.1
m was 46.2 % lower than AFS-I. At the 0.1-0.2 m layer, AFS-R showed the highest values of AC, 60 and
128.6 % higher than AFS-I and CT, respectively. However, at 0.1-0.2 m layer, AFS-I and CT did not differ
statistically from each other. No differences were found among the treatments at 0.2-0.4 m.

Considering the microbiological attributes (Table 4), significantly higher values of MBC were detected
under AFS-R, followed by AFS-I and CT. In contrast, no differences in BR were found among the
treatments. qMic values in AFS-R were twice higher than in AFS-I and in CT, that not differed statistically
from each other. AFS-R also showed the lowest qCO2 values, differing statistically from AFS-I and CT.
SOC content at the 0-0.10 m was higher in CT, followed by AFS-R and AFS-I.

Table 4. Soil microbiological attributes at the 0-0.1 m soil layer of an agroforestry-row (AFS-R),

agroforestry inter-row (AFS-I), and conventional tillage (CT).

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 Soil attributes AFS-R AFS-I CT P-value CV (%)

MBCa (mg C kg-1 soil) 278.61 a 218.01 b 167.27 c <0.01 12.64

BR (mg C-CO2 kg-1 soil h-1) 2.09 1.88 2.02 0.53 7.72

qMic (%) 1.34 a 0.67 b 0.70 b <0.01 10.20

qCO2 (mgC-CO2 g-1 MB-C h-1) 5.15 a 12.18 b 9.50 b <0.01 11.26

SOC (g kg-1) 27.35 b 24.88 c 31.22 a <0.01 5.79

NFB (CFU g-1 soil) 1.6 × 10 3 b 2.7 × 10 3 a 6.7 × 10 2 c <0.01 16.90

AB (CFU g-1 soil) 4.8 × 10 6 a 2.03 × 10 6 c 3.9 × 10 6 b <0.01 13.09

NOB (CFU g-1 soil) 4.02 × 10 6 4.5 × 10 6 3.8 × 10 6 0.17 12.33

Means followed by different letters in line are significantly different (Tukey, P ≤0.05)
a
Soil microbial biomass (MBC), basal respiration (BR), microbial quotient (qMic), metabolic

quotient (qCO2), soil organic carbon (SOC), nitrogen-fixing bacteria (NFB), ammonifying

bacteria (AB), nitrite-oxidizing bacteria (NOB).

The higher NFB was observed under AFS-I, followed by AFS-R and CT. AFS-R presented the highest values
of AB, followed by CT and AFS-I. No differences in NOB were observed among the treatments.

The PCA clearly separated the areas (Figure 3). PC 1 explained about 38% of the total variation and
distinguished AFS-I from CT, while PC 2 accounted for approximately 30.9% of the total variation and
separated AFS-R from AFS-I and CT. AFS-R was associated with macroporosity, bioporosity, saturated
hydraulic conductivity, microbial biomass carbon and ammonia-oxidizing bacteria. Fewer variables were
associated with AFS-I including bulk density, nitrogen-fixing and nitrite-oxidizing bacteria. Microporosity,
field capacity, mean weight diameter, and soil organic carbon were related to CT.

MBC, AB, Kfs and BR showed a strong positive correlation and were negatively correlated with BD,
demonstrating that increases in soil bulk density might impact the soil microbial community and its
activity, as well as soil water conductivity. Higher MWD values were associated with higher SOC content.

Discussion
Studies have shown that AFS contributes to the maintenance and enhancement of soil attributes related
to soil quality (Cezar et al., 2015; Cherubin et al., 2019; Froufe et al., 2020; Stöcker et al., 2020) since soil
management, especially tillage intensity and crop diversity, promotes changes in soil structural attributes,
which also influences the chemical and biological soil properties (Kim et al., 2020; Nunes et al., 2020).
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 AFS-R presented higher TP and lower BD at the superficial layer, an increased volume of pores
with large diameter (Ma and Bp) through the soil profile, as well as higher MDW, large and medium
macroaggregates (0.1-0.2 m) and a better overall VESS score, which are indicators of increased physical
quality. The absence of tillage and the action of the different root systems and soil organisms provided
by the diversity of plant species cultivated in the rows, as well as the plant residues added to ASF-R
through the pruning practice, may have contributed to these results (Cherubin et al., 2019; Rodríguez et al.,
2021). It would also explain some of the physical attributes showing better values in AFS-R compared to
the CT area tilled one time after fallow.

On the other hand, the vegetable cropping in agroforestry inter-rows increased soil BD (0-0.1 m) and
reduced soil TP (0-0.1 m), Ma and Bp (0-0.2 m) in relation to the row with trees. The TP decrease
observed in the surface layer from AFS-R to AFS-I (Table 2) can be attributed to the tillage employed for
vegetable cultivation in the inter-rows. Tillage disrupts the bonds between particles in the large
macroaggregates (responsible for soil structure stability), rearranging them inside the larger pores (ex.
Macropores), with a consequent reduction in TP and an increase in BD (Cavalcanti et al., 2020). This
hypothesis can be confirmed by the distribution of aggregate size classes, in which AFS-I presented a
reduction in the proportion of macroaggregates and, consequently, an increased number of
microaggregates.

Overall, the values of CT soil attributes remained between the AFS treatments. Many of the physical
variables in CT were not statistically different from AFS-R, probably due to the short time since the
conversion of the area from fallow to agriculture. Moreover, studies have shown that in the short term,
tillage increases soil porosity and aeration. Over the years, however, tillage practices end up in physical
quality degradation (Haruna et al., 2018; Vizioli et al., 2021), as has occurred in AFS-I.

Most of the significant differences between the areas occurred within the 0-0.2 m soil layer, probably due
to this depth being more prone to the rotary tiller action to prepare the horticultural soil beds. A rotary tiller
has blades that shred, mix and crumble the soil, breaking soil aggregates excessively and usually forming
a subsoil pan at the depth where implement weight is supported (Laudicina et al., 2017). This fact
supports our findings for MDW, aggregates size distribution, and VESS Sq among the studied areas, as
well as the values of macroporosity below the ideal limits for gaseous exchange and root growth (0.1 m3
m-3) (Xu et al., 1992) at the 0.1-0.2 m soil layer in AFS-I and CT.

Soil organic carbon did not vary in the more superficial layers, independent of the soil management
practices. SOC increases are usually observed in systems with positive carbon balance at the long term.
Thus, assessing the effects of vegetable cultivation on agroforestry inter-rows should be carried out over
longer periods. This is highlighted by the fact that in conventional tillage systems (i) SOC might be
redistributed within the soil profile (McCarty et al., 1998; Ferreira et al., 2018) and (ii) the formation and
stability of soil aggregates might be compromised by exposing the physically protected carbon to the soil
microbial community (Hok et al., 2021). Soil microbial activity increases due to the aeration and the

Page 11/23
availability of substrates for their metabolisms, and as result SOC is released from the system as CO2
(Xiao et al., 2019).

The visual evaluation of soil structure (VESS) demonstrated that soil structure was best following the
order AFS-R > AFS-I > CT. AFS-R presented roots and small aggregates in the whole block indicating
homogeneity of the soil structure. Less uniformity was observed between AFS-I and CT blocks, with all
the sampled points showing two layers of soil with structural differences: a superficial layer visually
structureless, consisting with loose soil particles with absence of aggregates, and another layer with
firmer, less porous and with bigger aggregates. These differences in the soil profile are characteristic from
anthropogenic management, in this case, mainly by the action of mechanization techniques as the rotary
tiller, which tends to disrupt soil structure and result in higher Sq values (Guimarães et al., 2017; Cherubin
et al., 2019).

The lower Sq found for CT might be an outcome from the poorer Sq attributed to its second soil layer,
probably remaining from the previous soil management explored in the area before the fallow period.
This finding reinforces the importance of plants root systems for mitigating compaction problems and
building soil structure (Zhang and Peng, 2021), considering the lower tillage mobilization in CT, but also
the absence of plants with aggressive root systems cultivated in the recent years compared to AFS-I.
Despite the differences, all managements where within the range of good soil quality based on the
interpretation that Sq < 3 is adequate and means that no management interventions should take place
(Ball et al., 2007; Guimarães et al., 2011).

The better structural attributes found in AFS-R may explain its highest AC and Kfs values. The
non-mobilization of the soil and the action of roots and edaphic fauna in the tree rows contributed to the
formation of large pores (Ma and Bp), through which the movement of air and water mainly occurs
(Borges et al., 2019). In AFS-I, pores size continuity may have been impaired due to soil tillage intensity,
resulting in lower AC and Kfs values (Dal Ferro et al., 2014; Borges et al., 2019), similarly to the findings of
Moraes et al. (2016) which observed doubled values for Kfs in no-till (NT) compared to CT and attributed
the results to the higher number and continuity of biopores present in the NT area.

The Kfs in CT was statistically similar to the AFS row and inter-row, probably due to soil tillage practice
with plowing and harrowing that, in the short term, reduces Bd, increases soil aeration (Haruna et al.,
2018), and the volume of cracks (Lopez-Bellido et al., 2016), implying in higher saturated hydraulic
conductivity in the CT area. Besides the lack of differences compared to AFS-R, it is important to
remember that the CT area presented critical values (lesser than 0.1 m3 m-3) for Ma at the 0.1-0.2 layer
(Xu et al., 1992), where water infiltration in the soil may be compromised.

PCA showed that the differences in soil management and crop diversity separated the areas regarding
soil physical and microbiological quality. Besides, it was evident that soil structure affects soil microbial
attributes. Soil aeration and water content are important parameters for plant growth and have also direct
effects on soil microbial community (Hungria et al., 2009; Silva et al., 2014), which might explain the

Page 12/23
negative relationship between BD and Kfs, and MBC. The better structure in AFS-R soil together with the
diversified vegetation and mulching may have contributed to the generation of a more beneficial
environment for the soil microbial community, being the tree rows more linked to MB-C and qMic. Higher
values of qMic suggest more favorable conditions for microbial biomass formation (Babujia et al., 2010),
with soil microorganisms increasing the proportion of carbon in its cells in relation to the SOC. On the
other hand, AFS-I and CT were more associated with qCO2, indicating a lower metabolic efficiency, and
more intense biological activity for carbon degradation than microbial biomass growth, which might
occur in stressful conditions (Bini et al., 2014; Lopes et al., 2021).

These results agree with a range of studies that have shown that conservationist soil management
systems provide higher MB-C values and lower RB values, resulting in higher qMic and lower qCO2
values (Hungria et al., 2009; Silva et al., 2014; Bini et al., 2014; Lopes et al., 2021). In this sense, the
absence of differences in BR might have a different interpretation for the areas, revealing a more stable
community in AFS-R, and a more stressed community in AFS-I and CT.

In AFS-I, the intense tillage may have promoted a rapid access of the microorganisms to SOC through
aggregates breakage and internal carbon exposition along the time, what ended up in the depletion of
energy resources and microbial community starving periods (Fiedler et al., 2016). For CT, although SOC
was the highest among the areas, the microorganisms were also in a stressful condition, which could be
explained by the low organic inputs added to the area and the presence of SOC content in large
aggregates (higher MWD values), protected from MB access.

Regarding microbiological attributes linked to N cycling, the higher AB in AFS-R can be an indicative that
in the rows, where the input of organic residues is higher and the root system is persistent, ammonium
production results from the mineralization of organic matter, and this process acts as an important
source of nitrogen to the trees (Beule et al., 2020). On the other hand, BNF was higher in AFS-I what might
be a surprising result if we take in account that many studies have shown that conservation soil practices
improve BNF compared to tillage (Torabian et al., 2019). However, this finding could be explained by the
possible differences in the diversity and the metabolism of the microbial communities in the areas. In
environments with more diverse C inputs, as native forests and perhaps AFS-R, microbial communities
are usually more diverse and formed by K-strategists, that are less metabolic active, grows slowly and are
more adapted to use C sources (Zhou et al., 2018). However, in more simplified and disturbed
environments like agricultural sites and in this case AFS-I, r-strategists may dominate, being less
generalists in terms of C sources, more active, and growing at higher rates, especially when there is great
availability of energy sources (Zhou et al., 2018).

Despite the absence of differences in NOB among the areas, PCA showed that this variable was more
associated with AFS-I, possibly due to tillage that mixes soil and disperse soil nitrogen and nitrifiers more
uniformly, creating more substrate-rich microsites for NOB development (Liu et al., 2017). NOB was also
negatively correlated with SOC, which could be expected since this study focused on autotrophic
nitrification (Liu et al., 2017). Once again, CT presented values that can be interpreted as in between the

Page 13/23
AFS-R and AFS-I areas, showing that with time tillage practices can degrade the soil and highlighting the
importance of adequate soil management strategies for achieving soil physical and biological quality in
agroecosystems.

Conclusions
Soil structural and biological attributes evaluated in this study indicate a better soil physical and
biological quality under the agroforestry tree rows. The lower plant diversity and tillage contributed to
structural and microbiological degradation in the first-year conventional tillage area and more intensively
in the four years agroforestry inter-row. These results indicate that more sustainable soil practices, like
reduced or no-tillage, should be studied and adopted as alternatives for cropping vegetable crops in
agroforestry systems.

Declarations
Acknowledgments

This study was suported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
(Capes) - Finance Code 001. The authors would like to thank the farmers that allowed their farms to be
sampled and all the people involved in this study, from soil sampling to writing.

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Figures

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Figure 1

Proportion of large macroaggregates (L-MAgg: 8–19 mm), medium (M- MAgg: 2–8 mm) and small (S-
MAgg: 0.25–2 mm) and microaggregates (MicroAgg: 0.053–0.25 mm), obtained by wet sifting, from
layers 0–0.10 (a) and 0.10–0.20 m (b) of an agroforestry-row (AFS-R), agroforestry inter-row (AFS-I), and
conventional tillage (CT). Means followed by the same letter within the same class of aggregates do not
differ by Tukey's test (P <0.05).

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Figure 2

VESS scores (Sq) for the first and second layers identified by the method (a) and overall Sq for total layer
(0–25 cm) (b) of an agroforestry-row (AFS-R), agroforestry inter-row (AFS-I), and conventional tillage
(CT). The gray lines indicate the VESS score (Sq = 3) considered as a threshold for root growth. Means
within each soil layer followed by the same letter do not differ by Tukey's test (P <0.05).

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Figure 3

Principal component analysis (PCA) based on structural and microbiological attributes assessed in the
agroforestry-row (AFS-R), agroforestry inter-row (AFS-I), and conventional tillage (CT) areas at the 0-0.1 m
soil layer. BD = bulk density; Bp = bioporosity; Ma = macroporosity; Mi = microporosity; FC = field
capacity; MWD = mean weight diameter; Kfs = saturated hydraulic conductivity; SOC = soil organic
carbon; MBC= microbial biomass carbon; BR = basal respiration; NFB = nitrogen-fixing bacteria; AB =
Ammonifying bacteria; NOB = nitrite-oxidizing bacteria.

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