J. Plant Nutr. Soil Sci. 2020, 000, 1–10 DOI: 10.1002/jpln.

201900456 1

Arbuscular mycorrhizal fungi and soil aggregation in a no-tillage system

with crop rotation
Mara Regina Moitinho1*, Carolina Fernandes2, Priscila Viviane Truber2, Adolfo Valente Marcelo3, José
Eduardo Corá2, and Elton da Silva Bicalho2
1 Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Rua Giuseppe
Máximo Scolfaro, 10000, 13083-970, Campinas, São Paulo, Brazil
2 UNESP—São Paulo State University, FCAV—School of Agricultural and Veterinary Studies, Via de Acesso Prof. Paulo Donato Castellane,

s/n, 14883292, Jaboticabal, São Paulo, Brazil

3 Rio Preto University Center (UNIRP), Rua Ivete Gabriel Atique, 45, 15025-400, São José do Rio Preto, São Paulo, Brazil

Abstract
Crop rotation adoption in no-tillage systems (NTS) has been recommended to increase the bio-
logical activity and soil aggregation, suppress soil and plant pathogens, and increase the produc-
tivity aiming at the sustainability of agricultural areas. In this context, this study aimed to assess
the effect of crop rotation on the arbuscular mycorrhizal fungi (AMF) community and soil aggre-
gation in a soil cultivated for nine years under NTS. Treatments consisted of combinations of
three summer crop sequences and seven winter crops. Summer crop sequences consisted of
corn (Zea mays L.) monoculture, soybean (Glycine max L. Merrill) monoculture, and soybean–
corn rotation. Winter crops consisted of corn, sorghum (Sorghum bicolor (L.) Moench), sunflower
(Helianthus annuus L.), sunn hemp (Crotalaria juncea L.), pigeon pea (Cajanus cajan (L.)
Millsp.), oilseed radish (Raphanus sativus L.), and millet (Pennisetum americanum (L.) Leeke).
Soil samples were collected at a depth of 0–0.10 m for analyses of soil chemical, physical, and
biological attributes. Spore abundance, total glomalin, and soil aggregate stability index were
higher in the soil under corn monoculture. The highest values of aggregate mean weight di-
ameter were observed in the soybean–corn rotation (3.78 mm) and corn monoculture (3.70 mm),
both differing from soybean monoculture (3.15 mm), while winter crops showed significant differ-
ences only between sorghum (3.96 mm) and pigeon pea (3.25 mm). Two processes were identi-
fied in the soil under summer crop sequences. The first process was observed in PC1 (spore
abundance, total glomalin, easily extractable glomalin, pH, P, and Mg2+) and was related to AMF;
the second process occurred in PC2 (aggregate mean weight diameter, soil aggregate stability
index, K+, and organic matter) and was related to soil aggregation. The nine-year no-tillage sys-
tem under the same crop rotation adoption influenced AMF abundance in the soil, especially
with corn cultivation in the summer crop sequence, which promoted an increased total external
mycelium length and number of spores of AMF. In addition, it favored an increased soil organic
matter content, which is directly related to the formation and stability of soil aggregates in these
managements.

Key words: glomalin / grasses / hyphae / legumes / NTS

Accepted May 20, 2020

1 Introduction
Increasing the efficiency of soil management practices by in-
tegrating and/or replacing more sustainable alternatives are
currently considered major challenges for the new directions
of global agriculture in order to achieve a sustainable and
profitable agricultural production while protecting the environ-
ment (FAO, 2015). In this context, Brazil is globally important
for food security as it is one of the largest producers and ex-
porters of important crops such as sugar and grains (soybean
and corn), among others. Brazilian grain production is esti-
mated to reach 236.7 million tons in the 2018/2019 season
(Conab, 2019). Brazil also has tremendous potential for
adopting the no-tillage system (NTS). Over 100 million hec-
tares worldwide are estimated to be under NTS, with approxi-
mately 32 million in Brazil (Motter and Almeida, 2015).
This system aims at a less soil mobilization with more crop
residues on the soil surface, resulting in improvements of soil
physical, chemical, and biological attributes in addition to ena-
bling loss reduction via erosion, increased nutrient cycling
rate, and high soil organic matter accumulation (Pinheiro
et al., 2004; dos Santos et al., 2011; Higo et al., 2016; Wang
et al., 2016), as well as providing favorable conditions for
increased soil microorganism community activity such as
arbuscular mycorrhizal fungi (AMF). AMF are obligatory

* Correspondence: M. R. Moitinho; e-mail: maramoitinho@gmail.com

ª 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

2 Moitinho, Fernandes, Truber, Marcelo, Corá, da Silva Bicalho
biotrophic organisms that establish a mutualistic symbiosis
mostly with terrestrial plant roots, working as an expansion of
the plant–root system, providing higher water and nutrient
absorption (Borie et al., 2006) and hence favoring gains in
production. The presence of plants with mycorrhizal depend-
ence stimulates the establishment of these fungi in the soil
(Gosling et al., 2016), and the beneficial effects of this associ-
ation can be extended over time, which leads to significant
improvements in production systems (Orio et al., 2016).
The conservationist soil management based on crop diversifi-
cation is an appropriate alternative to maintain or increase
soil AMF species diversity (Wang et al., 2016; de Pontes
et al., 2017). These fungi, in addition to improving water and
nutrient absorption by plants, may favor soil aggregation pro-
cesses by the action of AMF mycelia that surround soil par-
ticles, providing high soil aggregate stability (Rillig et al.,
2003; Wu et al., 2014; Xie et al., 2015).
Furthermore, AMF hyphae, when associated with plant roots,
can form a net which includes soil unit particles, allowing the
formation of new aggregates in the soil (Wendling et al.,
2005; Orio et al., 2016). Soil aggregation also occurs through
the chemical action of a glycoprotein produced by fungi,
known as glomalin (Wu et al., 2014), which can be found in
the spore walls and AMF hyphae and may accumulate glyco-
protein in the soil after their decomposition. As the glomalin
has adherent and cementing properties, its presence can
positively affect aggregate formation by uniting soil particles
(Purin and Klauberg Filho, 2010) and increase the stability of
the aggregate already formed (Xie et al., 2015).
In this context, the assessment of certain soil chemical attrib-
utes, such as organic matter, pH, and phosphorus available
for plants, is also very important when assessing soil struc-
ture, since they influence the formation of organomineral
complexes and the diversity and dynamics of soil microbial
activity (Lal, 2009; de Pontes et al., 2017). In fact, several
studies have focused on NTS related to improvements in soil
properties. However, unlike our study, most of them have as-
sessed systems established for less than eight years, usually
using only crop rotation and being recommended for increas-
ing the biological activity and soil aggregation (Pinheiro et al.,
2004; Higo et al., 2016; Orio et al., 2016).
This study is based on the hypothesis that the interaction
between cover crops and AMF community and hence soil ag-
gregation is related to the intrinsic characteristics of each spe-
cies and management of crop residues, besides the edapho-
climatic conditions of each region. Also, the time of adoption
of the system with the same crop sequences can affect the
dynamics of nutrient release, also influencing the dynamics of
these interactions. Therefore, this study aimed to assess the
effect of crop rotation on the AMF community and soil aggre-
gation in a soil cultivated for nine years under NTS.
ª 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Plant Nutr. Soil Sci. 2020, 000, 1–10
2 Material and methods
2.1 Characterization of the study areas and
treatments
The experiment was conducted at an experimental field lo-
cated in the São Paulo State University, School of Agricultural
and Veterinary Studies (UNESP/FCAV), Jaboticabal, SP, Bra-
zil, at 595 m asl and geographical coordinates 21°15¢22¢¢ S
and 48°18¢58¢¢ W. The regional climate is classified as
B1rB¢4a¢ according to the Thornthwaite (1948) system, indi-
cating a humid mesothermal region with little water deficit and
average temperatures of the warmest and coldest months
higher than 22 and 18°C, respectively, in addition to an
average annual precipitation of 1,425 mm. The soil is classi-
fied as a very clayey Ferralsol (Santos et al., 2013), wavy re-
lief, and particle size distribution of sand (2.0–0.05 mm), silt
(0.05–0.002 mm), and clay (< 0.002 mm) fractions of 370, 65,
and 565 g kg–1, respectively, at a depth of 0–0.20 m. This
area has been used for soybean and corn production under
conventional tillage system for 25 years.
An experiment was carried in September 2002 under NTS to
assess the effect of crop sequences on soil attributes. Before
experiment set up, a subsoiling operation was conducted at a
depth of 0.40 m, and 1.5 Mg ha–1 of limestone with 100% of
total neutralizing power was incorporated into the soil using
plowing and harrowing operations to increase base saturation
to 70%. The experimental design was a strip-block design
with three replications. Treatments consisted of the combina-
tion of three summer crop sequences and seven winter crops
(cover crops), totaling 21 plots per experimental block. Each
plot corresponded to an area of 200 m2 (20 · 10 m).
Summer crop sequences consisted of corn monoculture
(CC), soybean monoculture (CS), and soybean–corn rotation
(SCR) successively cultivated over the years, being sown in
October. Corn was cultivated in the area when this study was
carried out in the 2010/2011 season. Winter crops (cover
crops) consisted of corn, sorghum, sunflower, sunn hemp,
pigeon pea, oilseed radish, and millet, which were sown from
February to March. The same winter crop was cultivated at
the same plot each agricultural season. Winter crops were
chopped with a straw chopper at the full flowering of sunn
hemp, pigeon pea, oilseed radish, and millet, and after har-
vesting corn, sorghum, and sunflower. The results presented
in this study are from the data collected in the 2010/2011 sea-
son, the ninth agricultural year after the experiment was set
up.
2.2 Cultivation and crop management
In all seasons, corn and sunflower cultivated in the
winter were mechanically sown at an interrow spacing of
0.90 m aiming at population densities of 55,000 and
88,000 plants ha–1, respectively. The other winter crops were
mechanically sown at an interrow spacing of 0.45 m aiming at
population densities of 555,000 plants ha–1 for oilseed radish
(Siletina cultivar) and sunn hemp (common cultivar),
665,000 plants ha–1 for millet (BN-2 cultivar) and pigeon pea
(dwarf cultivar), and 175,000 plants ha–1 for sorghum (1G150
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J. Plant Nutr. Soil Sci. 2020, 000, 1–10
cultivar). Soybean and corn were mechanically sown in the
summer without prior soil tillage. An early-cycle soybean culti-
var was sown at an interrow spacing of 0.45 m aiming at
reaching a population density of 480,000 plants ha–1. Soy-
bean seeds were inoculated with Bradyrhizobium japonicum.
A corn hybrid with a super-early cycle was sown at an inter-
row spacing of 0.90 m aiming at reaching a population density
of 66,000 plants ha–1.
Topdressing fertilization was conducted for corn when plants
presented six completely unfolded leaves by applying
100 kg ha–1 of N as ammonium sulfate. Fertilization recom-
mendations for corn and soybean in the summer were based
on the results of soil chemical analyses from the previous
growing season assessments and considering high-expected
yields (Raij et al., 1997). Winter crops received no fertilizers.
2.3 Assessment of soil properties
Composite and disturbed soil samples were collected in Octo-
ber 2011 from three points per plot at a depth of 0.0–0.1 m be-
fore sowing the summer crops in the 2011/2012 season,
when the soil was covered with crop residues. The following
biological attributes were determined from these samples: to-
tal external mycelium length (TEML), using the direct quantifi-
cation method of extraradical mycelium, which is based on
the use of filtration membrane by induced fluorescence meth-
ods with fluorescein diacetate (FDA) hydrolysis (Melloni and
Cardoso, 1999) and the simplified equation of Newman
(1966), correcting the obtained value according to the water
content in the substrate and aliquot volumes [Eq. (1)]; spore
abundance (SA), using the wet sieving technique and centri-
fugation in water and sucrose (Colozzi Filho and Balota,
1994); and easily extractable glomalin (EEG) and total gloma-
lin (TG) (Rillig et al., 2003).
L ¼ 0:21387·n=10  U; (1)
in which L is the extraradical mycelium length (m g–1 dry sub-
strate), n is the number of intersections of hyphae with hori-
zontal lines of the gridline, and U is the amount of water in
10 g of the moist substrate (g).
In short, EEG was determined by extraction in an autoclave,
using 1 g of soil sample and 8 mL of 20 mM sodium citrate so-
lution (pH 7.4) and autoclaving at 121°C for 30 min. TG was
obtained using 1 g of soil sample and 8 mL of 50 mM sodium
citrate (pH 8.0) and autoclaving at 121°C for 60 min. It took
more than one autoclaving cycle (three to 13 cycles depend-
ing on the sample) to extract this fraction until the sample
reached a light yellow color.
The aggregate stability index (ASI) and aggregate mean
weight diameter (AMWD) were determined according to Nim-
mo and Perkins (2002). ASI was obtained using 4 g of aggre-
gates with a diameter of 1–2 mm, which were stirred for 3 min
in a vertical oscillation apparatus, with an oscillation ampli-
tude of 1.3 cm and rotation of 35 rpm. On the other hand,
AMWD was determined using 25 g of aggregates with diame-
ters of 3–4 and 6 mm. These samples passed through six
sieves (4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 mm) inside a verti-
ª 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
AMF and soil aggregation in a no-tillage system 3
cal oscillation apparatus. The aggregates were immersed in
water and received light stirring in the vertical direction for
10 min. Subsequently, they were stirred for another 10 min
and placed in an oven at 105°C for 24 h.
For soil chemical characterization, the active acidity (pH in
CaCl2 0.01 mol L–1) was determined potentiometrically using
the soil to CaCl2 ratio of 1:2.5, while the potential acidity
(H++Al3+) was determined according to Raij et al. (2001). Ex-
changeable calcium (Ca2+), magnesium (Mg2+), and potas-
sium (K+) and available phosphorus (P) were extracted using
the ion exchange resin method proposed by Raij et al. (2001).
The sum of bases (SB), cation exchange capacity (CEC), and
base saturation (BS) were calculated based on the results of
soil chemical analyses. Organic matter (OM) was quantified
by multiplying the total organic matter (TOC) by 1.724. TOC
was determined by oxidizing a 100-mg soil sample at 900°C
in a Shimadzu Total Organic Carbon Analyzer TOC–V
coupled on a Shimadzu Solid Sample Module SSM-5000A. A
calibration curve with standard potassium biftalate was ob-
tained for the calculations.
2.4 Data processing and analysis
Initially, the data were submitted to analysis of variance
(F-test) considering the strip-block design with three replica-
tions. The mean comparison was performed by the Tukey’s
test at 5 and 1% probability. After variable standardization
(zero mean and unit variance), the data were submitted to the
multivariate exploratory analysis of principal components. Co-
efficients of linear functions, which define the principal com-
ponents, were used in interpreting their significance, consid-
ering their signal and relative size as an indication of the
weight to be assigned to each variable. Only coefficients with
high values were considered for interpretation, usually those
higher than or equal to 0.50 in absolute value. The statistical
analyses were carried out using the software SAS 9.1 (SAS
Institute, Cary, NC, USA) and Statistica 7.0 (StatSoft Inc., Tul-
sa, OK, USA).
3 Results
3.1 Variation of soil attributes under summer and
winter sequences
The analysis of variance indicated a significant effect of the
interaction between summer and winter crop sequences
(F = 25.6; p < 0.01) on total external mycelium length (TEML),
indicating that these sequences interact with each other and
influence fungi development (Tab. 1). Thus, the interaction
was sliced to verify the associated effect of sequences on
TEML (Tab. 2). The highest TEML values were observed in
the sequences with CC (corn monoculture) in the summer
and corn (6.57 m g–1 soil) or millet (6.67 m g–1 soil) in the
winter, the latter also with SCR (soybean–corn rotation)
(5.84 m g–1 soil) in the summer. On the other hand, the lowest
TEML mean value was observed for SCR in the summer and
sunn hemp in the winter (1.86 g–1 soil); the same was ob-
served for pigeon pea in the winter for the sequences SCR
(2.42 g–1 soil) and CC (2.51 g–1 soil) in the summer.
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4 Moitinho, Fernandes, Truber, Marcelo, Corá, da Silva Bicalho J. Plant Nutr. Soil Sci. 2020, 000, 1–10

Table 1: Total external mycelium length (TEML), spore abundance (SA), easily extractable glomalin (EEG), and total glomalin (TG) at a depth
of 0–0.1 m after the cultivation of summer crop sequences and winter crops for nine years under the no-tillage system.a

TEML SA EEG TG
Summer crop sequences (S)
(m g–1 soil) (spores 50 mL–1 soil) (mg g–1 soil)

Soybean–corn rotation 3.94 1.95 b 1.03 3.23 b

Corn monoculture 4.47 3.38 a 1.09 3.77 a

Soybean monoculture 3.85 2.00 b 1.04 3.44 b

F (summer crop sequences) 9.18* 9.17** 0.41NS 16.8*

CV (%) 12.4 33.2 20.1 8.72

TEML SA EEG TG
Winter crops (W)
(m g–1 soil) (spores 50 mL–1 soil) (mg g–1 soil)

Corn 5.37 2.56 ab 1.04 3.46

Sunflower 3.81 3.11 ab 1.02 3.30

Oilseed radish 3.78 2.11 ab 1.03 3.33

Millet 5.45 1.89 b 1.07 3.64

Pigeon pea 3.14 1.78 b 1.07 3.55

Sorghum 4.45 4.00 a 1.06 3.57

Sunn hemp 2.60 1.67 b 1.07 2.51

F (winter crops) 38.1** 4.32** 0.96NS 2.53NS

CV (%) 12.7 45.50 6.54 6.66

F (S · W) 25.6** 1.08NS 0.77NS 1.13NS

CV (%) 9.79 31.3 8.20 5.76

a NS = not significant, * = significant at 5%, and ** = significant at 1%. CV = coefficient of variation. Means followed by different letters in the col-

umns differ from each other by the Tukey’s test at 5% probability.

Table 2: Total external mycelium length (TEML, in m g–1 soil) at a depth of 0.0–0.1 m as a function fered from that observed in SCR
of summer crop sequences and winter crops after nine years under the no-tillage system.a (1.95 spores 50 mL–1 soil), but it did
not differ from CS (soybean mono-
Winter crops Summer crop sequences culture) (2.0 spores 50 mL–1 soil) re-
Soybean–corn rotation Corn monoculture Soybean monoculture
gardless of the winter crop used
(Tab. 1). Regarding winter crops,
Corn 4.04 Cc 6.57 Aa 5.51 Ab the highest mean values for SA
Sunflower 3.51 CDb 4.73 Ba 3.19 CDb
were observed for grasses, such as
sorghum (4.00 spores 50 mL–1 soil)
Oilseed radish 5.49 ABa 3.61 BCDb 2.26 Dc and corn (2.56 spores 50 mL–1 soil),
Millet 5.84 Aa 6.67 Aa 3.83 BCb legumes, such as oilseed radish
(2.11 spores 50 mL–1 soil), and
Pigeon pea 2.42 DEb 2.51 Db 4.49 Aba oleaginous, such as sunflower
Sorghum 4.43 BCa 4.09 BCa 4.82 Aba (3.11 spores 50 mL–1 soil), with no
differences among them. However,
Sunn hemp 1.86 Eb 3.11 CDa 2.82 CDa sorghum differed from sunn hemp
(1.67 spores 50 mL–1 soil), pigeon
aMeans followed by different uppercase letters in the columns and lowercase letters in the rows pea (1.78 spores 50 mL–1 soil), and
differ from each other by the Tukey’s test at 5% probability. millet (1.89 spores 50 mL–1 soil),
regardless of the summer crop
Significances were observed for spore abundance (SA) in (Tab. 1).
summer (F = 9.17, p < 0.01) and winter sequences (F = 4.32,
p < 0.01), indicating that the number of spores presents Crop sequences in NTS did not affect the amount of easily
distinct patterns when these sequences are analyzed in- extractable glomalin (EEG) determined in the 0–0.1 m soil
dividually. The mean SA in CC (3.38 spores 50 mL–1 soil) dif- layer (Tab. 1). The total glomalin (TG) value was higher in the

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J. Plant Nutr. Soil Sci. 2020, 000, 1–10 AMF and soil aggregation in a no-tillage system 5

area with CC (3.77 mg g–1 soil), differing from areas with CS Soil chemical attributes presented significant effects only for
(3.44 mg g–1 soil) and SCR (3.23 mg g–1 soil), regardless of summer sequences, except for Mg, which had no significant
the winter crop, as the analysis of variance showed no signifi- effects for sequences and interactions (Tab. 4). In this sense,
cant effect (F = 2.53, p > 0.05) when analyzing the winter regarding soil organic matter (OM), CC presented a mean val-
crops, indicating that TG values have a similar variation pat- ue (26.07 g dm–3) higher than CS (23.88 g dm–3), with no dif-
tern when the seven crops are compared. ference between SCR and the sequences CC and CS. The
lowest mean value for soil pH was observed in CC (4.91), dif-
The aggregate stability index (ASI) showed a significant effect fering from SCR (5.27) and CS (5.19). The soil under CS
(F = 3.28, p < 0.05) only on summer sequences. The highest showed higher P values (41.19 mg dm–3), differing from SCR
values were observed in CC (63.09%) and SCR (60.38%). (30.81 mg dm–3) and CC (25.5 mg dm–3), which did not differ
Continuous sequences presented significant differences from each other. On the contrary, the lowest mean K value
among them, with the lowest mean value observed in CS was observed in CS (3.32 mmolc dm–3), differing from SCR
(59.76%) (Tab. 3). The aggregate mean weight diameter (4.12 mmolc dm–3) and CC (4.38 mmolc dm–3). The ex-
(AMWD) had a significant effect on the summer (F = 27.5, changeable calcium was the only soil attribute that differed
p < 0.01) and winter sequences (F = 3.59, p < 0.05) (Tab. 3). significantly in the three assessed sequences, with values of
Therefore, AMWD presents a distinct behavior when analyz- 21.24, 25.17, and 31.57 mmolc dm–3 for CC, CS, and SCR,
ing these two sequences alone. The highest AMWD values respectively.
were observed in SCR (3.78 mm) and CC (3.70 mm), both dif-
fering from CS (3.15 mm), which presented the lowest value Moreover, H+Al showed the highest value under the
for this attribute. Regarding the winter sequence, a difference CC sequence (41.10 mmolc dm–3), differing from SCR
was observed only between sorghum (3.96 mm) and pigeon (32.57 mmolc dm–3) and CS (32.05 mmolc dm–3). On the con-
pea (3.25 mm) (Tab. 3). trary, base saturation (BS) had the lowest value under CC
(48.50%), differing from the other sequences. The sum of
bases (SB) showed the highest value under the SCR se-
Table 3: Soil aggregate stability index (ASI) and aggregate mean quence (50.81 mmolc dm–3), while SCR and CS differed from
weight diameter (AMWD) at a depth of 0.0–0.1 m after the cultivation each other regarding the attribute cation exchange capacity
of summer crop sequences and winter crops for nine years under the
(CEC), with mean values of 83.38 and 75.44 mmolc dm–3, re-
no-tillage system.a
spectively.
ASI AMWD
Summer crop sequences (S)
(%) (mm) 3.2 Relationship between soil attributes and
summer and winter sequences
Soybean–corn rotation 60.38 ab 3.78 a
The biplot (Fig. 1), generated with the first two principal com-
Corn monoculture 63.09 a 3.70 a
ponents, accounted for 67.61% of the total variance of soil
Soybean monoculture 59.76 b 3.15 b attributes, with 46.18% of the variance retained in PC1 and
21.43% in PC2. The correlations between each attribute and
F (summer crop sequences) 3.28* 27.5**
its respective principal components are shown in Tab. 5. The
CV (%) 6.97 8.40 most relevant attributes in PC1 were pH (–0.92), TG (0.75),
ASI AMWD
Winter crops (W)
(%) (mm)

Corn 61.00 3.52 ab

Sunflower 61.55 3.59 ab

Oilseed radish 58.77 3.42 ab

Millet 61.66 3.57 ab

Pigeon pea 60.88 3.25 b

Sorghum 62.44 3.96 a

Sunn hemp 61.22 3.50 ab

F (winter crops) 0.41 NS 3.59*

CV (%) 8.72 11.7

F (S · W) 0.87 NS 1.27NS
Figure 1: Biplot graph containing soil properties and the assessed
CV (%) 6.88 9.00
sequences. AMWD = aggregate mean weight diameter; ASI = aggre-
gate stability index; SA = spore abundance; EEG = easily extractable
a NS = not significant and ** = significant at 1%. CV = coefficient of
glomalin; TG = total glomalin; OM = organic matter; K+ = exchange-
variation. Means followed by different letters in the columns differ able potassium; P = phosphorus available for plants; and Mg2+ =
from each other by the Tukey’s test at 5% probability. exchangeable magnesium.

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6 Moitinho, Fernandes, Truber, Marcelo, Corá, da Silva Bicalho J. Plant Nutr. Soil Sci. 2020, 000, 1–10

Table 4: Organic matter (OM), soil pH, available phosphorus (P), exchangeable potassium (K+), calcium (Ca2+), and magnesium (Mg2+),
potential acidity (H++Al3+), sum of bases (SB), cation exchange capacity (CEC), and base saturation (BS) at a depth of 0.0–0.1 m after the culti-
vation of summer crop sequences and winter crops for nine years under the no-tillage system.a

OM pH P K+ Ca2+
Summer crop sequences (S)
(g kg–1) (CaCl2) (mg dm–3) (mmolc dm–3)
Soybean–corn rotation 25.31 ab 5.27 a 30.81 b 4.12 a 31.57 a
Corn monoculture 26.07 a 4.91 b 25.52 b 4.38 a 21.24 b
Soybean monoculture 23.88 b 5.19 a 41.19 a 3.32 b 25.17 c
F (summer crop sequences) 3.65* 19.41** 9.48** 6.96** 24.45**
CV (%) 9.37 4.31 38.09 24.87 23.04
OM pH P K+ Ca2+
Winter crops (W)
(g kg–1) (CaCl2) (mg dm–3) (mmolc dm–3)
Corn 25.06 5.17 26.28 3.07 26.50
Sunflower 24.17 5.28 33.28 4.07 28.39
Oilseed radish 24.94 5.17 36.33 4.53 28.28
Millet 23.94 5.12 35.56 4.27 24.67
Pigeon pea 25.67 5.11 33.39 3.82 25.44
Sorghum 25.67 5.01 29.72 3.85 23.22
Sunn hemp 26.17 5.02 33.00 3.97 25.44
F (winter crops) 0.84NS 1.99NS 0.77NS 2.08NS 1.36NS
CV (%) 10.87 4.92 40.03 26.12 24.33
F (S · W) 0.87NS 0.67NS 0.75NS 0.28NS 0.58NS
CV (%) 10.02 4.52 39.76 25.08 23.89
Mg2+ H++Al3+ SB CEC BS
Summer crop sequences (S)
(mmolc dm–3) (%)
Soybean–corn rotation 15.12 32.57 b 50.81 a 83.38 a 60.36 a
Corn monoculture 13.55 41.10 a 39.16 b 80.21 ab 48.50 b
Soybean monoculture 14.91 32.05 b 43.39 b 75.44 b 57.17 a
F (summer crop sequences) 2.11NS 16.81** 12.71* 5.56** 18.78**
CV (%) 17.03 17.89 18.67 7.89 13.87
Mg2+ H++Al3+ SB CEC BS
Winter crops (W)
(mmolc dm–3) (%)
Corn 15.94 33.72 45.51 79.23 57.39
Sunflower 16.56 30.44 49.01 79.45 60.67
Oilseed radish 13.11 34.28 45.92 80.20 56.72
Millet 15.00 34.17 43.94 78.11 55.78
Pigeon pea 13.94 36.83 43.21 79.93 53.67
Sorghum 13.50 37.89 40.57 78.46 51.17
Sunn hemp 13.61 39.33 43.02 82.36 52.00
F (winter crops) 2.20NS 2.50NS 1.12NS 0.29NS 2.35NS
CV (%) 19.29 20.20 19.51 9.36 15.12
F (S · W) 0.67NS 0.57NS 0.59NS 0.19NS 0.58NS
CV (%) 18.54 18.34 19.01 8.87 13.90

a NS= not significant and ** = significant at 1%. CV = coefficient of variation. Means followed by different letters in the columns differ from each
other by the Tukey’s test at 5% probability.

ª 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

J. Plant Nutr. Soil Sci. 2020, 000, 1–10 AMF and soil aggregation in a no-tillage system 7

Table 5: Correlation between soil attributes and each principal com- system and the presence of thin roots in these crops, which
ponent (PC1 and PC2). enable easier colonization by fungal hyphae (Orio et al.,
2016). When associated with plant roots, fungi mycelium act
Principal component PC1 PC2 exploring a large volume of soil that would not be accessible
Explained variance (%) 46.18a 21.43a
to the roots if there were no such associations. The high soil
exploration by roots favors an increase in water and nutrient
Correlation absorption by plants, as hyphae act as extensions of the root
Aggregate mean weight 0.21 –0.69
system (Xie et al., 2015; Wang et al., 2016). The results of
diameter this study corroborate those observed by Borie et al. (2006),
who also showed the beneficial effect of grasses on TEML.
Aggregate stability index 0.16 –0.57 Thus, there is an evident relationship between external myce-
Spore abundance 0.61 –0.13 lium and roots and their impact on host growth. Plant growth
can vary with the proportion between internal and external
Easily extractable glomalin 0.66 –0.18 fungal mycelium. Negative responses (parasitism) may result
Total glomalin 0.75 –0.20 from large root colonization by AMF and little external growth.
On the contrary, parasitism may also occur when there are lit-
pH –0.92 –0.12 tle root colonization and large production of external myce-
Organic matter 0.31 –0.62 lium (Wu et al., 2014; de Pontes et al., 2017).
Exchangeable potassium 0.23 –0.55
The results of spore abundance (SA) indicated that crop rota-
Available phosphorus –0.69 0.19 tion in NTS influences AMF abundance in the soil, mainly
under grass cultivation. In this sense, high sporulation is re-
Exchangeable magnesium –0.74 0.25
lated to plants with an abundant root system and fast growth,
Interpretation Attributes associ- Attributes asso- with increased contact between roots and AMF propagules
ated with ciated with soil and high ability to provide photosynthates to the fungus, as in
mycorrhizal fungi aggregation the root system of grasses (Daniels Hetrick and Bloom,
1986). Moreover, the increase only in the total glomalin (TG)
aPercentage of variation of the original dataset retained by its princi- content, but not in the easily extractable glomalin (EEG), after
pal component. Correlations in bold (> 0.50 in absolute value) were summer crop sequences (Tab. 1) may be related to an in-
considered in the interpretation of the principal component. crease in TG in the soil over the years in the experimental
area. It may have occurred because TG can be more adhered
to soil aggregates, thus being considered more recalcitrant,
Mg (–0.74), P (–0.69), EEG (0.66), and SN (0.61), while
while EEG is the recently produced glomalin, being more la-
AMWD (–0.69), OM (–0.62), ASI (–0.57), and K (–0.55) were
bile in the soil (Purin and Klauberg Filho, 2010). CC provided
the most relevant attributes for PC2 (Tab. 5).
higher glomalin production among summer crops (Tab. 1). TG
According to the relationships observed in PC1 (Fig. 1) be- values were low despite the summer crop that preceded soil
tween CC and CS and their respective sets of variables in the sampling in SCR was also corn, indicating that the use of
same direction, the soil under CC presented the highest SA, grass in continuous sequences favored high glomalin produc-
TG, and EEG values and the lowest pH, P, and Mg values. tion compared to the grass in rotation with legumes, demon-
The opposite behavior was observed for CS, i.e., this system strating cumulative effect over the cultivation years.
is characterized by presenting high pH, P, and Mg values and
Only summer crops presented a significant effect on soil
low SA, TG, and EEG values (Fig. 1). In PC2, the attributes
chemical attributes, except for Mg (Tab. 4). The attributes
AMWD, OM, ASI, and K are more associated with SCR and
OM, K, and CEC were benefited by the corn crop in CC or
CC (with a small representation) (Fig. 1).
SCR, while pH and BS were benefited by CS or SCR. How-
Thus, considering the attributes retained in PC1 and PC2 and ever, the attributes that showed the highest values when as-
that principal components are independent and orthogonal to sessed only in continuous sequences were P (CS) and H+Al
each other, two distinct processes occur in the soil under (CC), being the attributes Ca and SB benefited when as-
summer sequences: the first process occurs in PC1 and is re- sessed in SCR. In short, corn cultivated as a monoculture
lated to the mycorrhizal fungi, and the other process occurs in (CC) or in rotation (SCR) under NTS favored important chemi-
PC2 and is related to soil aggregation. cal characteristics related to soil fertility, such as high values
of OM, K+, Ca2+, CEC, and SB, in addition to a balanced pH.

4 Discussion One of the main characteristics of NTS is a reduction in the

4.1 Effect of summer and winter crop sequences
on mycorrhizal fungi and soil attributes
Tables 1 and 2 show that grasses (corn and millet) promoted
an increase in total external mycelium length (TEML) in the
soil. This fact is related to the greater abundance of the root
OM oxidation rate, which is a good strategy to improve soil
physical and chemical attributes, mainly through increases in
cation exchange capacity and acidity correction by organic-
chemical processes (Lal, 2009). In this context, while promot-
ing an increase in the pH-dependent cation exchange ca-
pacity, OM benefits the adsorption of exchangeable cations
(Ca2+, Mg2+, and K+) by exchanges with H+ of organic

ª 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

8 Moitinho, Fernandes, Truber, Marcelo, Corá, da Silva Bicalho
functional groups (Watson et al., 2002). Moreover, the bio-
mass with the highest C/N ratio has the highest aggregating
effect due to a slower decomposition and formation of inter-
mediate organic compounds that contribute to increasing OM
content in the soil. Therefore, grasses such as corn act more
effectively to promote the formation of aggregates both by the
direct action of the roots and by the supply of more permanent
and stable organic residues (Xie et al., 2015).
4.2 Effect of summer and winter crop sequences
on soil aggregation
The aggregate stability index (ASI) and aggregate mean
weight diameter (AMWD) were high in the summer sequen-
ces CC and SCR (Tab. 3). These results demonstrate the
positive effect of the corn root system on soil aggregation,
being important to mention that corn was the summer crop
that preceded soil sampling in SCR. The role of roots, espe-
cially of grasses, has been vital in forming and stabilizing soil
aggregates. These beneficial effects can be attributed mainly
to their root system, which can act promoting the approxima-
tion of soil particles, in addition to the renewal of the root sys-
tem and uniform distribution of exudates, which stimulate
microbial activity, whose by-products affect the aggregate for-
mation and stabilization (Xie et al., 2015).
Accordingly, Wendling et al. (2005) assessed the aggregate
stability of an Oxisol and observed that tifton grass is an ap-
propriate option for the formation and stabilization of soil ag-
gregates. Also, the authors found an AMWD of 2.09 mm in
the soil under grass cultivation, which was very close to the
AMWD found in the soil under the native forest (2.46 mm).
Pinheiro et al. (2004) also found a beneficial action of the root
system of grasses on aggregates of an Oxisol. These authors
found AMWD values of 4.02 mm in the soil under grass culti-
vation, 3 mm under the no-tillage system, and 2 mm under
the conventional tillage.
In this study, among the winter crops, sorghum and pigeon
pea provided an aggregate formation with the highest and
lowest AMWD values, respectively (Tab. 3). Despite assess-
ing different winter crops, the use of NTS provided the same
physical management during the agricultural seasons, with
the same number of operations in the field, which explains
the small variation in AMWD (from 3.25 to 3.96 mm). How-
ever, these values found in plots cultivated in the winter with
pigeon pea (AMWD = 3.25 mm) and sorghum (AMWD =
3.96 mm) are probably due to differences in the use of winter
crops with purposes of green manure for pigeon pea and
grain production for sorghum. In this case, pigeon pea was
chopped during the full flowering, which was reached in about
50–60 days after sowing in this region. For sorghum, whose
crop residues were chopped after harvesting, physiological
maturity and harvest occurred at about 110 days after sowing.
Consequently, after nine years of cycling these crop residues
in the winter, the longer permanence of sorghum develop-
ment in the field favored the formation of large soil aggregates
compared to the soil under pigeon pea.
ª 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Plant Nutr. Soil Sci. 2020, 000, 1–10
4.3 Interdependence relationships between soil
attributes and management
The direct relationship between spore abundance, total glo-
malin, and easily extractable glomalin (CC) (Fig. 1, Tab. 5)
was already expected, being often discussed in the literature
and usually associated with AMF abundance (Rillig et al.,
2003; Borie et al., 2006; Purin and Klauberg Filho, 2010; Wu
et al., 2014; Xie et al., 2015), as glomalin is produced by AMF
(Wu et al., 2014). However, the antagonistic relationship ob-
served in the biplot for pH and P (CS) as a function of the at-
tributes associated with AMF abundance (spore abundance,
total glomalin, and easily extractable glomalin) (Fig. 1) are
possibly related to the fact that soil pH can affect the quantity
and diversity of communities of mycorrhizal fungi (Moebius-
Clune et al., 2013). Fungi have their development favored in
environments with pH lower than 5. Availability and toxicity of
mineral nutrients are also related to soil pH (Primavesi,
2006). In this context, there is evidence that lower soil pH val-
ues in the CC sequence (4.91) may be contributing to higher
SA, TG, and EEG values in this system.
Corroborating this study, some authors have found a negative
correlation between AMF mycelia and available P content in
the soil (Nóbrega et al., 2001; Watson et al., 2002), suggest-
ing that high P contents may cause an inhibitory effect on fun-
gal propagules in the rhizosphere, which leads to a reduced
colonization by self-regulatory mechanisms of symbiosis
(Moreira and Siqueira, 2006). Phosphorus in plants also con-
trols the degree of root colonization by AMF and affects the
development of internal and external hyphae. The addition of
low P doses in soils with limited or very low P availability in-
creases both colonization and external mycelium because
when P limits root development, it also limits fungus develop-
ment (Nóbrega et al., 2001).
Various studies have reported the beneficial effect of glomalin
on the aggregation and stability of soil aggregates (Purin and
Klauberg Filho, 2010; Wu et al., 2014; Xie et al., 2015). How-
ever, in this study, TG and EEG were retained in PC1, while
the attributes related to soil aggregation (ASI, OM, AMWD,
and K) were retained in PC2 (Fig. 1, Tab. 5). Considering that
PCs are orthogonal to each other, the attributes retained in
PC1 act independently from those retained in PC2. Similar re-
sults have been found in other studies. Borie et al. (2006)
found no correlation between aggregate stability indices and
the amount of glomalin in the soil, even after 20 years of culti-
vation in an Ultisol. These authors suggested that aggregate
formation and stabilization in this soil might be related to fac-
tors other than AMF. Rillig et al. (2003) assessed differences
in hyphae length, glomalin, and soil aggregate stability in
semiarid regions of Spain and also found no positive correla-
tion between the amount of glomalin and the aggregate stabil-
ity index. According to these authors, the results suggest that
glomalin was not the main binding agent of aggregates in this
soil, which is considered rich in carbonate. Moreover, this re-
sult may also be due to the high aggregation level found in
these soils.
Our results also indicate that other factors, such as OM, are
acting in the formation and stabilization of soil aggregates. As
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J. Plant Nutr. Soil Sci. 2020, 000, 1–10
shown in Fig. 1, the attributes ASI, OM, AMWD, and K, re-
tained in PC2, are projected at the same direction and are
more representative in the SCR sequence, i.e., there was
high dispersion of the SCR sequence points towards the
same direction where these attributes are projected (Fig. 1,
Tab. 5).
The direct relationship of the attributes retained in PC2 may
occur because OM acts as a soil cementing agent, and an in-
crease in aggregate stability is closely associated with the
OM capacity in adhering to soil mineral particles (Lal, 2009;
de Pontes et al., 2017). In addition, soils managed under
NTS favor a high organic matter accumulation and hence
high formation and stability of soil aggregates, which is re-
lated to high additions of organic material associated with the
absence of soil disturbance (Lal, 2009). Furthermore, there is
a relationship between the physical protection of soil carbon
and the decomposition action of microorganisms provided by
soil aggregates, especially in clayey Oxisols (dos Santos
et al., 2011).
The location of these attributes directed towards the area of
SCR indicates that there may be a high soil aggregation and
organic matter contribution in this sequence, and possible
combined effect of crops (grasses and legumes). This behav-
ior was identified in the multivariate analysis of the data
(Fig. 1), while the univariate analysis showed that the behav-
ior of these attributes was the same for CC and SCR (Tabs. 3
and 4). Therefore, the roots of these plants under rotation
may be acting more effectively in the formation of soil aggre-
gates compared to CC and CS sequences.
5 Conclusions
The no-tillage system with crop rotation adoption influenced
arbuscular mycorrhizal fungi abundance in the soil, especially
after corn cultivation in the summer, promoting an increased
total external mycelium length and number of spores, as well
as a high total glomalin production.
The summer crop sequences soybean–corn rotation and
corn monoculture favored an increased soil organic matter
content after nine years under the same system, which is di-
rectly related to aggregate formation and stability. Also, the
sequence with soybean provided a high available phosphorus
content.
Acknowledgments
We thank the Graduate Program in Soil Science of the
School of Agricultural and Veterinary Studies of the São Pau-
lo State University (FCAV/UNESP), the Coordination for the
Improvement of Higher Education Personnel (CAPES) for the
financial support, and the reviewers and editor, who contrib-
uted to the quality of this manuscript.
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