Nativa, Sinop, v. 10, n. 3, p. 410-416, 2022.
Pesquisas Agrárias e Ambientais
DOI: https://doi.org/10.31413/nativa.v10i3.13792 ISSN: 2318-7670
Silicon application and mycorrhiza inoculation promoted
Leucaena leucocephala
growth in a soil highly contaminated by manganese
Juliette Freitas do CARMO1, Kaio Gráculo Vieira GARCIA1*, Paulo Furtado MENDES FILHO1,
Arthur Prudêncio de Araújo PEREIRA1, José Israel PINHEIRO1
1Federal University of Ceará, Fortaleza, CE, Brazil.
E-mail: kaiovieira@ufc.br
ORCID: (0000-0003-3619-9456; 0000-0003-4980-5136; 0000-0001-7030-6206; 0000-0001-9402-3243; 0000-0002-0665-3892)
Submitted on 2022/05/04; Accepted on 2022/09/05; Published on 2022/09/19.
ABSTRACT: Arbuscular mycorrhizal fungi (AMF) can increase the acquisition of silicon (Si) and, therefore,
alleviate the problems caused by metallic toxicity in plants, but this effect remains poorly understood. The
objective was to evaluate the influence of Si application on the growth of Leucaena leucocephala inoculated with
AMF (Claroideoglomus etunicatum) in a soil contaminated by manganese (Mn). We exposed plants to increasing
levels of Si (0, 100, 200 and 400 mg kg-1) in the soil for 90 days. Intermediate levels of Si and AMF inoculation
significantly increased shoot and root dry mass, the number of sheets, root system length and mycorrhizal
colonization. The abundance of AMF spores decreased linearly with increasing levels of Si applied to the soil,
suggesting a low correlation with mycorrhizal colonization. In addition to the higher Mn contents in the shoots
and, mainly, in the roots, the combined application of Si and inoculation with AMF significantly reduced foliar
toxicity by more than 40%, when compared to the absence of Si and AMF inoculation. Our results
demonstrated a synergistic effect of AMF and Si in improving the growth and tolerance of L. leucocephala plants
in soil contaminated by Mn.
Keywords: heavy metals; stress; remediation; AMF; Mn toxicity.
Aplicação de silício e inoculação de micorriza promove crescimento de
Leucaena leucocephala
em solo altamente contaminado por manganês
RESUMO: Os fungos micorrízicos arbusculares (FMA) podem aumentar a aquisição de silício (Si) e, portanto,
amenizar os problemas causados pela toxidez metálica nas plantas, mas esse efeito ainda é pouco conhecido. O
objetivo foi avaliar a influência da aplicação de Si no crescimento de Leucaena leucocephala inoculada com FMA
(Claroideoglomus etunicatum) em solo contaminado por manganês (Mn). Expusemos as plantas a níveis crescentes
de Si (0, 100, 200 e 400 mg kg-1) no solo por 90 dias. Níveis intermediários de Si e inoculação de FMA
aumentaram significativamente a massa seca da parte aérea e radicular, o número de folhas, o comprimento do
sistema radicular e a colonização micorrízica. A abundância de esporos de FMA diminuiu linearmente com o
aumento dos níveis de Si aplicados ao solo, sugerindo uma baixa correlação com a colonização micorrízica.
Além dos maiores teores de Mn na parte aérea e, principalmente, nas raízes, a aplicação combinada de Si e
inoculação com FMA reduziu significativamente a toxidez foliar em mais de 40%, quando comparada à ausência
de Si e inoculação de FMA. Nossos resultados demonstraram um efeito sinérgico de FMA e Si na melhoria do
crescimento e tolerância de plantas de L. leucocephala em solo contaminado por Mn.
Palavras-chave: metais pesados; estresse; remediação; FMA; toxidez por Mn.
1. INTRODUCTION
Mining is one of the oldest productive activities of
humanity. Brazil is one of the largest producers and exporters
of mining products, with an enormous mineral heritage.
According to the Brazilian Mining Institute/IBRAM (2020),
mining activity represents 4% of the country's Gross
Domestic Product, being one of the pillars of Brazil's
economic support. However, despite its economic
importance, income generation and employment, factors that
drive the country's growth, mining can cause environmental
damage and constitute an imminent risk to human life
(HADDAWAY et al., 2019).
Manganese (Mn), despite being an essential micronutrient
for all living organisms (TANG et al., 2021), presents a highly
toxic reaction when present in high concentrations (LI et al.,
2019). In plants, Mn toxicity can change metabolic processes
and, consequently, limit their growth and development (LI et
al., 2019). The most visible effects of plant exposure to high
Mn concentrations in the soil are, in general, described by
brown spots, chlorosis and leaf necrosis, which can limit the
photosynthetic efficiency (FARIA et al., 2020; GARCIA et
al., 2020). In humans, excessive Mn exposure can damage to
the nervous system and intellectual deficit in children
(BUDINGER et al., 2021).
In mining areas, Mn can reach high concentrations
through the deposition of tailings residues, which can release
large amounts of Mn into the soil (HUANG et al., 2014).
These conditions, in addition to the low soil fertility
Carmo et al.
Nativa, Sinop, v. 10, n. 3, p. 410-416, 2022.
411
commonly found in these areas, make the establishment of
plant species an important challenge. In this sense, strategies
that mitigate this impact and improve soil revegetation are
extremely necessary.
A viable alternative for the revegetation contaminated soil
is to stimulate the plant association with beneficial soil
microorganisms, such as arbuscular mycorrhizal fungi (AMF)
(GARCIA et al., 2020). The use of leguminous plants has
been recommended in the literature, mainly due to the
association with nitrogen-fixing bacteria and mycorrhiza,
attributes that increases soil quality and the potential of plant
survival (BALIEIRO et al., 2017).
AMF are able to associate symbiotically with more than
80% of terrestrial plants (YOU et al., 2021). This association
can increase nutrient absorption, water, attenuate the
negative effects of heavy metals (HM) and promote plant
growth in environments under contamination
(SPAGNOLETTI et al., 2017). It is known that AMF can
protect plants against HM through several mechanisms,
including the retention and immobilization of these elements
in roots of colonized plants (SPAGNOLETTI et al., 2017),
chelation through glomalin synthesis (VODNIK et al., 2008)
and accumulation of metals on the surface of AMF-spores
(GARCIA et al., 2020). In addition, they can increase the
absorption of other elements present in the soil solution,
such as silicon (Si), a beneficial micronutrient, which can also
mitigate the stress caused by HM in plants (ZEHRA et al.,
2020).
Mycorrhizal plants often show greater tolerance to excess
Mn in the soil (GARCIA et al., 2020). In this sense, evidence
suggests that one of the reasons for the attenuation of Mn
toxicity in the presence of AMF is due to the increase in the
absorption of Si, which in turn may be involved in mitigating
the adverse effects of metals in plants metabolism, especially
Mn (HORST; MARSCHNER, 1978). Although some studies
report the effects of Si on the attenuation of toxicity by HM
in plants, to date, there are no publications on the role of Si,
as well as a possible synergistic effect between Si and AMF
in mitigating Mn toxicity in L. leucocephala.
In our study, we tested the effect of Si doses and AMF
inoculation on the initial development of L. leucocephala
cultivated in a soil degraded by Mn mining. Our hypothesis
was that the interaction between Si and mycorrhiza
inoculation attenuates phytotoxicity by Mn and promotes
better plant growth conditions, thereby increasing its
efficiency in the revegetation process.
2. MATERIAL AND METHODS
2.1. Localization area and experimental soil
The experiment was conducted in a greenhouse, located
at the Fortaleza, Ceará, Brazil. For plants growth, we
collected soil at a depth of 0-20cm in a manganese mining
area located in Ocara, Ceará, Brazil. Subsequently, the soil
was sieved with the aid of a 2mm sieve and then autoclaved
at 121ºC and 1 atm of pressure for 2h, to eliminate fungal
propagules and other existing microorganisms. Soil chemical
characterization analysis (Table 1) was made according to the
methods described by Teixeira et al. (2017).
Briefly, the pH was measured in water (1:2.5) by
potentiometry; exchangeable aluminum (Al3+), was extracted
with 1M KCl solution and determined by titration; Ca2+ and
Mg2+ were extracted with 1M KCl solution and determined
by atomic absorption spectrometry; Potential acidity (H+Al),
was extracted with calcium acetate buffered (pH 7.0) and
determined by titration; Phosphorus (P), sodium (Na+) and
potassium (K+) were extracted with Mehlich 1 solution, with
P determined by colorimetry and K+ and Na+ by flame
photometry; N was determined by the semimicro Kjeldahl
method; Mn, Fe, Cu, Zn, were extracted with Mehlich's
solution 1 and determined by atomic absorption
spectrometry. The Si was extracted with 0.2 mol L-1
ammonium acid oxalate solution (pH 3.0) and determined by
ICP-OES.
Table 1. Soil chemical characterization collected at the manganese
mining area, Ocara, CE.
Tabela 1. Caracterização química do solo coletado na área de
mineração de manganês, Ocara, CE.
pH (Water) 6.7
Al3+
- - - - -(cmol
c
kg
-1
)
- - - - -
0.1
Ca2+ 5.7
Mg2+ 2.08
Na+ 0.04
K+ 0.35
H+Al 0.9
P (mg kg-1) 6.8
N (g kg-1) 1.25
Si 0.00
Mn
(mg kg
-1
)
332
Fe 5.15
Cu 1.82
Zn 5.23
2.2. Cultivated plant and AMF inoculum
The plant species chosen in our study was Leucaena
leucocephala. We chose this species because it is to be of
multiple use and because it has potential for use in
revegetation practices in soils contaminated by HM (JUSON
et al., 2016). In addition, this species is able to grow well in
soils with low fertility, probably due to its ability to fix
atmospheric nitrogen and to associate with AMF, thus
making it a promising plant species. The AMF used was the
species Claroideoglomus etunicatum. To obtain the inoculum soil,
we used corn plants (Zea mays L.) as trap crop, cultivated in
sterile sand. We chose this AMF species for its good
performance in increasing plant growth in soil contaminated
by Mn (GARCIA et al., 2020). A volume of 40g inoculum,
containing spores, fragments of colonized roots, mycelium
and sand, were used as AMF inoculum soil. We performed a
preliminary count of spores in the inoculum soil, which
showed ~300 viable spores in 40g soil.
2.3. Experimental setup and plant growth conditions
The experiment consisted of a completely randomized
design, in a 4x2 factorial scheme, with four replications.
Thus, our study consisted of four doses of Si (0; 100; 200;
400 mg kg-1) and two mycorrhizal treatments (inoculated
with Claroideoglomus etunicatum and control–non-inoculated).
The parameters mycorrhizal colonization and abundance of
AMF spores in the soil were analyzed without the presence
of the uninoculated treatment (control), since such plants did
not show evidence of colonization by AMF, as well as no
Silicon application and mycorrhiza inoculation promoted Leucaena leucocephala growth in a soil highly contaminated by manganese
Nativa, Sinop, v. 10, n. 3, p. 410-416, 2022.
412
spores were observed in their rhizospheres, confirming the
effectiveness of the adopted sterilization process.
The soil was distributed in plastic pots with a capacity of
1.5 liters, using 1 kg of soil per pot. Soil fertility correction
was performed by applying 10 mg kg-1 of N, 15 mg kg-1 of P,
100 mg kg-1 of K and 3 mg kg-1, mixed with the substrate.
The sources of nutrients used were CO(NH2)2, P2O5, KCl
and CaSO4, respectively for nitrogen, phosphorus, potassium
and calcium. One day after correction, Si doses (0, 100, 200,
and 400 mg kg-1) were applied in the pots containing the soil,
in the form of sodium silicate (Na2SiO3). The doses applied
were chosen following the methodology proposed by Garg
and Singh (2018).
Leucena seeds were superficially disinfected in 95%
alcohol. Subsequently, they were heated to a temperature of
80ºC for 5 minutes, in order to break dormancy. The
production of seedlings to be used in our study took place in
plastic trays, by placing one seed per cell at a depth of 2 cm.
The substrate used to produce the seedlings was sterilized
sand.
At 14 days after sowing (DAS), the plants were
transplanted into the pots. Inoculation with AMF occurred
during the transplant process. Soil moisture in the
experimental trial was maintained at approximately 60% of
the soil's water holding capacity and plants were allowed to
grow for 90 days after transplanting (DAT).
2.4. Measurements and analytical determinations
At the end of the experimental conduction, growth data
were measured evaluating shoot and root dry mass (g),
number of sheets (plant-1 unit) and root length (cm). To
obtain the shoot dry mass, the plants freshly harvested were
immediately dried in an oven with forced air circulation at a
temperature of around 65ºC. The number of sheets was
obtained through direct counting and the root length was
measured from the plant neck to the tip of the main root,
using a millimeter ruler. After obtaining the dry mass of
shoots and roots, the material was ground to determine Mn
levels in shoots and roots. Mn contents were determined by
atomic absorption spectrophotometry after digestion of
HNO3-HClO4 (TEIXEIRA et al., 2017).
Arbuscular mycorrhizal colonization was determined
following the methodological procedures described by
Phillips and Hayman (1970). The root fragments were
washed in running water and, later, the root cortex was
clarified by heating (60 ºC) in a water bath in a 10% KOH
solution. Then the, roots were stained with 5% acidified
Parker® pen ink (VIERHEILIG et al., 1998). AMF spore
abundance in soil was determined by extracting 100 g of soil
through wet sieving (GERDEMANN; NICOLSON, 1963).
Leaf toxicity by Mn was performed by counting the
number of sheets with symptoms and calculated according to
the formula: (number of sheets with symptoms / total
number of sheets) x 100 (GARCIA et al., 2020).
2.5. Statistical analysis
Data set was submitted to analysis of variance (ANOVA),
using the F test. When significant differences were observed,
the quantitative data were adjusted with fitted with regression
models and the qualitative data were compared using the
Scott-Knott test, using the software Sisvar 5.6 (FERREIRA,
2011).
3. RESULTS
3.1. Plant growth
All plants that received silicon doses above 200 mg kg-1
significantly reduced shoot dry mass production (Figure 1a).
On the other hand, when they were inoculated with AMF,
they showed an increase in the production of shoot dry mass,
when compared to plants without inoculation, mainly in the
presence of Si, regardless of the applied dose. A similar
pattern was found for root dry mass. However, plants
without AMF inoculation showed a significant reduction,
even with the application of increasing doses of Si (Figure
1b).
The number of sheets of the inoculated treatments with
AMF was significantly (~362%) higher, especially when
compared to plants without AMF inoculation (Figure 1c).
The root length increased as a function of increasing doses
of Si applied in the soil in both treatments (with and without
AMF) (Figure 1d). However, the difference between
inoculated and non-inoculated treatments was only
evidenced at the dose of 200 mg kg-1 of Si, where plants
inoculated with AMF showed a significant increase of 15.7%
in root length when compared to plants without AMF.
Figure 1. Dry mass of shoot (a) and root (b) as a function of Si doses
and treatments with AMF inoculation. Number of sheets as a
function of treatment with AMF inoculation (c). Root length as a
function of Si doses and treatments with AMF inoculation (d).
Means followed by the same letters do not differ by Scott-Knott test
(p≤ 0.05).
Figura 1. Massa seca da parte aérea (a) e raíz (b) em função das doses
de Si e tratamentos com inoculação de FMA. Número de folhas em
função do tratamento com inoculação de FMA (c). Comprimento
da raiz em função das doses de Si e tratamentos com inoculação de
FMA (d). As médias seguidas pelas mesmas letras não diferem pelo
teste de Scott-Knott (p≤ 0,05).
3.2. Mycorrhizal Colonization and AMF Spore
Abundance in Soil
Mycorrhizal colonization showed a maximum increase
(~32%) up to the estimated dose of 181.54 mg kg-1 of Si,
which started to decrease from this value onwards (Figure
2a). On the other hand, the abundance of AMF spores in the
rhizosphere decreased as a function of the increasing doses
of Si (Figure 2b).
Si doses (mg kg
-1
)
0 100 200 300 400
Shoot dry mass (g)
0.0
0.5
1.0
1.5
2.0
2.5
- AMF y = -0.000003x
2
+0.0009x+0.27 R
2
=0.99
**
+AMF y = -0.000006x
2
+0.002x+1.80 R
2
=0,92
**
Si doses (mg kg
-1
)
0 100 200 300 400
Root dry mass (g)
0.0
0.2
0.4
0.6
0.8
1.0
-AMF y = -0.000264x+0.20 R
2
=0.97
**
+AMF y = -0.000004x
2
+0.001x+0.63 R
2
= 0.79
**
ab
d
c
Treatments with AMF inoculation
Number of shetts (plant
-1
unit)
0
2
4
6
8
10
12
- AMF
+ AMF
b
a
c
Si doses (mg kg
-1
)
0 100 200 300 400
Root length (cm)
20
22
24
26
28
30
32
34
36
-AMF y = 0.000098x
2
-0.021x+27.32 R
2
=0.99
**
+AMF y = -0.000051x
2
+0.053x+21.41 R
2
=0.91
**
d
Carmo et al.
Nativa, Sinop, v. 10, n. 3, p. 410-416, 2022.
413
Figure 2. Mycorrhizal Colonization (a) and AMF Spore Abundance
in Soil (b) in L. leucocephala as a function of the doses of Si applied
to the soil.
Figure 2. Colonização micorrízica (a) e abundância de esporos de
FMA no solo (b) em L. leucocephala em função das doses de Si
aplicadas no solo.
3.3. Manganese in shoots and roots
The levels of manganese in shoots of plants inoculated
with AMF showed a maximum increase (346.13 mg kg-1) up
to the estimated dose of 125.78 mg kg-1 of Si, which started
to decrease from this value onwards (Figure 3a). On the
treatment without AMF inoculation decreased linearly as a
function of increasing doses of Si (Figure 3a). All plants
inoculated with AMF showed higher manganese content in
shoots than plants without AMF.
In the roots, the Mn contents were higher than in the
shoot, mainly in plants inoculated with AMF (Figure 3b).
Inoculation with AMF provided a maximum increase
(2367.49 mg kg-1) in the manganese content in roots up to
the estimated dose of 88 mg kg-1 of Si, which started to
decrease from this value onwards, while the treatment
without AMF decreased linearly as a function of increasing
doses of Si (Figure 3b).
3.4. Leaf toxicity
Leaf toxicity showed a linear decrease as a function of
increasing doses of Si applied to the soil in both treatments
(with and without AMF) (Figura 4). However, the inoculation
of plants with AMF was able to significantly decrease the leaf
toxicity by Mn, when compared to plants without AMF, up
to the dose of 200 mg kg-1 of Si, while at the maximum dose
of this element (400 mg kg-1 of Si) there was no difference in
leaf toxicity when comparing plants with and without AMF
(Figure 4).
Figure 3. Manganese content in shoots (a) and roots (b) of L.
leucocephala as a function of Si doses and treatments with AMF
inoculation.
Figura 3. Teor de manganês na parte aérea (a) e nas raízes (b) de L.
leucocephala em função de doses de Si e tratamentos com inoculação
de FMA.
Figure 4. Leaf toxicity of L. leucocephala as a function of Si doses and
treatments with AMF inoculation.
Figura 4. Toxidez foliar de L. leucocephala em função de doses de Si
e tratamentos com inoculação de FMA
4. DISCUSSION
The results obtained point to a benefit generated by the
association of AMF with L. leucocephala, since, there was an
increase in the production of shoot dry mass, root dry mass
and number of sheets, suggestive of the participation of the
mycorrhizal fungus in the increase of Si absorption. It is
known that high concentrations of Mn in the soil can impair
plant growth (FARIA et al., 2020). It can be affirmed that
AMF increased the tolerance of L. leucocephala to the Mn
excess present in the soil, since, AMF in association with
Si doses (mg kg
-1
)
0 100 200 300 400
Mycorrhizal colonization (%)
10
15
20
25
30
35
+AMF y = -0.000280x
2
+0.101x+23.11 R
2
= 0.99
**
a
Si doses (mg kg
-1
)
0 100 200 300 400
AMF spore abundance in soil (100 g soil
-1
)
1100
1200
1300
1400
1500
1600
+AMF y = -1.008261x+1562.35 R
2
=0.98
**
b
Si doses (mg kg
-1
)
0 100 200 300 400
Manganese in shoots (mg kg
-1
)
0
100
200
300
400
500
+AMF y = -0.000871x
2
+0.219x+332.35 R
2
=0.87
*
-AMF y = -0.131693x+90.86 R
2
=0.74
**
a
Si doses (mg kg
-1
)
0 100 200 300 400
Manganese in roots (mg kg
-1
)
0
500
1000
1500
2000
2500
3000
+AMF y = -0.008081x
2
+1.420x+2367.49 R
2
=0.98
**
-AMF y = -3.42x+1928.94 R
2
=0.94
**
b
Si doses (mg kg
-1
)
0 100 200 300 400
Leaf toxicity (%)
0
10
20
30
40
50
60
-AMF y = -0.129734x+56.89 R
2
=0.97
**
+AMF y = -0.025104x+12.38 R
2
=0.88
**
Silicon application and mycorrhiza inoculation promoted Leucaena leucocephala growth in a soil highly contaminated by manganese
Nativa, Sinop, v. 10, n. 3, p. 410-416, 2022.
414
plants improve their nutritional status, including in relation
to Si, contributing to its growth and decreasing the availability
of some metals for plants, for example the Mn (GARG;
SINGH, 2018; GARCIA et al. 2020).
According to Emamverdian et al. (2018), Si can act on the
complexation of metal ions in the cell wall, decreasing, in
turn, the translocation of ions from the root to the shoot of
the plant. This fact may have contributed to the improvement
in the growth of L. leucocephala plants under Mn stress in the
present study. In the same way, Wang et al. (2020) also
observed that the application of Si in the soil increased the
growth of Brassica chinensis L. grown in soil contaminated with
several HM.
Regarding root length, there is little differentiation
between treatments (with and without AMF) and both
obtained positive results in relation to root system growth,
evidencing possible action of Si in increasing the tolerance of
plants in the presence of Mn. However, the mechanisms of
Si responsible for this possible protection are still not well
understood (VACULÍK et al., 2021). Ur Rahman et al. (2021)
state that Si supplementation in wheat plants grown under
cadmium stress increased the length and volume of their
roots.
It is worth mentioning that the relevant results obtained
with the addition of Si, in terms of benefits for plants, are
more evident under stress conditions (BHAT et al., 2019). It
is also important to note that, in addition to plants,
microorganisms are also affected when exposed to high levels
of HM in the soil. Such a fact can explain why, in this study,
inoculation with AMF did not have much influence on root
length, being possible that the high concentrations of Mn
have reduced the ability of the fungus to influence the greater
growth of these roots. Except for the root length, the results
found suggest that AMF inoculation and Si application in the
soil, act synergistically and alleviate Mn stress, increasing the
growth of L. leucocephala.
With regard to mycorrhizal colonization, it was observed
that, despite its decrease in plants under higher Si doses in
the soil (Figure 5), the symbiosis with C. etunicatum was
effective and did not limit the development of these plants.
Figure 5. Mycorrhizal colonization of L. leucocephala at different silicon doses applied to the soil (a – 0 mg kg-1; b – 100 mg kg-1; c – 200 mg
kg-1; d – 400 mg kg-1).
Figura 5. Colonização micorrízica de L. leucocephala em diferentes doses de silício aplicadas ao solo (a – 0 mg kg-1; b – 100 mg kg-1; c – 200
mg kg-1; d – 400 mg kg-1).
Moreover, the foliar symptoms of Mn toxicity did not
increase with the decrease in colonization, on the contrary,
there was a decrease in the percentage of foliar toxicity as the
Si doses were increased, indicating that there was no isolated
effect of AMF. Garg and Sing (2018) also point out that this
interaction does not decrease the ability of AMF to colonize
plant roots. It is worth mentioning, that another factor that
may be related to the decrease in mycorrhizal colonization is
the density of the roots, since decreases in the dry mass of
plants’ roots were also observed under higher Si doses.
Increasing Si doses significantly influenced AMF spore
abundance in soil, which decreased with increasing amounts
of applied Si in the soil. However, this factor was not
reflected in greater damage to the plants, since they all
showed similar development. Was noticed that mycorrhizal
colonization had a low relationship with spore abundance,
this is consistent with the fact that the number of spores in
the soil minimally impacts the potential for mycorrhizal
colonization in the roots (BAREA et al., 1991). The few
studies that evaluated the interaction between Si and
mycorrhiza in plants under stress conditions due to the
presence of large amounts of Mn did not correlate
mycorrhizal colonization with AMF spore abundance in soil
and with the influence of Si (YOST; FOX, 1982). In general,
what can be inferred from the results obtained is that Si had
a negative influence on AMF spore abundance in soil, since
increasing the applied doses resulted in a decrease in the
number of spores in the rhizosphere of L. leucocephala.
All plants inoculated with AMF showed higher levels of
Mn in the shoot than uninoculated plants, a result that differs
from some studies found with other HM (ULTRA et al.,
2021). Garcia et al. (2020) explain that often the uptake of
Mn and its concentrations in plants are lower in mycorrhizal
plants. Although not well understood, this behavior has been
attributed to different mechanisms of AMF protection to
plants against excess HM in the soil, including the retention
and immobilization of these elements on the surface of AMF
spores (GARCIA et al., 2020). The result for this study,
however, is not an indicator of inefficiency for the treatment
with mycorrhiza, since this association obtained the best
results for all the variables analyzed in this study. Even
though high levels of Mn were obtained in the shoots, the
concentration of this element did not exceed the level
considered toxic to plants, which is 400 mg kg-1 of Mn in the
dry mass of the shoot (KABATA-PENDIAS, 2010).
Moreover, it is also worth mentioning that Si can help
against the effects of HM contamination in plants. Among
the mechanisms that act in this process are the decrease of
Carmo et al.
Nativa, Sinop, v. 10, n. 3, p. 410-416, 2022.
415
metal ions in the soil and their absorption in the plant,
chelation of metals, gene regulation related to the transport
of metals, stimulation of antioxidants and structural changes
in plants (BHAT et al., 2019).
In general, in both treatments (with and without AMF)
the highest levels of Mn were found in the roots compared
to the shoot of the plants, with emphasis on the mycorrhizal
plants. This greater accumulation of Mn in the roots may
explain the lower levels found in the shoot, which may
characterize this plant species as a potential indication for use
in phytostabilization programs. Motaharpoor et al. (2019)
also observed high concentrations of Cd in the roots of
plants inoculated with AMF, having the authors attributed
these results to the sequestration of metals in the cell wall and
in compartments in the structures of the AMF. With respect
to Si, it is notable that this element, as well as AMF, can also
help in the retention of metals in the roots. According to
Khan et al. (2021), Si can help to increase the cell wall
thickness of roots, forming a physical barrier that binds and
restricts the transport of HM.
The treatments with AMF inoculation showed the lowest
leaf toxicity percentage in relation to the treatments without
inoculation. These results may be partially related to the
decrease in Mn content observed in the shoots of the plants
(Figure 3a) as a result of the interaction with Si. However, the
decrease in Mn content in the shoots of the plants is
apparently not related to the greater absorption by the roots
observed, since Mn concentrations in roots also tended to
decrease with increasing Si supplementation (Figure 3b).
The decrease in leaf toxicity percentage in these plants
may probably be associated with a decrease in the availability
of this metal in the soil and, consequently, its lower
absorption. Some authors recognize the action of Si in the
immobilization of HM in the soil (VACULÍK et al., 2021).
Bhat et al. (2019) explain that Si can stimulate plant roots to
release a greater amount of flavonoids and organic acids that
can act in the chelation of metals in the soil and, therefore,
reduce their phytotoxicity. Another effect of Si in the soil
would be to favor an increase in the pH of the soil solution,
resulting consequently in a decrease in the availability of
metallic elements (KHAN et al., 2021). Analyzing especially
the action of AMF, results similar to those found in this study
were observed by Garcia et al. (2020) while studying the
attenuation of foliar toxicity in mycorrhizal L. leucocephala
plants under increasing levels of Mn in the soil.
The interaction of Si doses with AMF for the leaf toxicity
variable was of great value, from the lowest to the highest
applied Si dose. It can be affirm that the association of AMF
C. etunicatum with L. leucocephala, in conjunction with Si,
significantly attenuates the foliar symptoms of toxicity caused
by Mn excess.
5. CONCLUSIONS
The inoculation of AMF and the application of Si in the
soil, together, alleviate the stress caused by Mn and increase
the growth of L. leucocephala. Furthermore, Si proved to be
effective, up to a dose of 200 mg kg-1, in maximizing the
growth of mycorrhizal plants under Mn stress. It is likely that
this dose of Si may differ depending on the type of soil, metal,
plant species, and AMF. In a future perspective, our study
can serve as a basis to assist in revegetation practices in
mining areas with Mn excess.
6. REFERENCES
BALIEIRO, F. de C.; COSTA, C. A.; de OLIVEIRA, R. B.;
de OLIVEIRA, R.; DONAGEMMA, G. K.; de
ANDRADE, A. G.; CAPECHE, C. L. Carbon stocks in
mined area reclaimed by leguminous trees and sludge.
Revista Arvore, v. 32, p. 1-10, 2017.
https://doi.org/10.1590/1806-90882017000600010
BAREA, J. M.; AZCÓN-AGUILAR, C.; OCAMPO, J. A.;
AZCÓN, R. Morfologia, anatomia y citologia de las
micorrizas vesiculo-arbusculares. In: BAREA, J. M. &
LIVARES, J. (Eds). Fijación y movilización biológica
de nutrientes, Madrid, p. 149-173, 1991.
BHAT, J. A.; SHIVARAJ, S. M.; SINGH, P.; NAVADAGI,
D. B.; TRIPATHI, D. K.; DASH, P. K.; SOLANKE, A.
U.; SONAH, H.; DESHMUKH, R. Role of silicon in
mitigation of heavy metal stresses in crop plants. Plants,
v. 8, p. 1-20, 2019.
https://doi.org/10.3390/plants8030071
BUDINGER, D.; BARRAL, S.; SOO, A. K. S.; KURIAN,
M. A. The role of manganese dysregulation in
neurological disease: emerging evidence. The Lancet
Neurology, v. 20, p. 956-968, 2021.
https://doi.org/10.1016/S1474-4422(21)00238-6
EMAMVERDIAN, A.; DING, Y.; XIE, Y.; SANGARI, S.
Silicon mechanisms to ameliorate heavy metal stress in
plants. BioMed Research International, v. 2018, p. 1-
10, 2018. DOI: https://doi.org/10.1155/2018/8492898
FARIA, J. M. S.; MARTINS, D.; PAULA, A.; BRITO, I.;
BARRULAS, P.; ALHO, L.; CARVALHO, M. Toxic
levels of manganese in an acidic Cambisol alters
antioxidant enzymes activity, element uptake and
subcellular distribution in Triticum aestivum.
Ecotoxicology and Environmental Safety, v. 193, p. 1-
9, 2020. https://doi.org/10.1016/j.ecoenv.2020.110355
FERREIRA, D. F. Sisvar: a computer statistical analysis
system. Ciência e Agrotecnologia, v. 35, p. 1039-1042,
2011. https://doi.org/10.1590/S1413-
70542011000600001
GARCIA, K. G. V., MENDES FILHO, P. F.; PINHEIRO,
J. I.; CARMO, J. F. do; PEREIRA, A. P. de A.;
MARTINS, C. M.; ABREU, M. G. P. de; OLIVEIRA
FILHO, J. de S. Attenuation of Manganese-Induced
Toxicity in Leucaena leucocephala Colonized by Arbuscular
Mycorrhizae. Water, Air and Soil Pollution, v. 231, p.
1-15, 2020. https://doi.org/10.1007/s11270-019-4381-9
GARG, N.; SINGH, S. Arbuscular Mycorrhiza Rhizophagus
irregularis and Silicon Modulate Growth, Proline
Biosynthesis and Yield in Cajanus cajan L. Millsp.
(pigeonpea) Genotypes Under Cadmium and Zinc Stress.
Journal of Plant Growth Regulation, v. 37, p. 46-63,
2018. https://doi.org/10.1007/s00344-017-9708-4
GERDEMANN, J. W.; NICOLSON, T. H. Spores of
mycorrhizae endogone species extracted from soil by wet
sieving and decanting. Transactive British Mycology
Society, v. 46, p. 235-244, 1963.
https://doi.org/10.1016/S0007-1536(63)80079-0
HUANG, J.; NARA, K.; ZONG, K.; WANG, J.; XUE, S.;
PENG, K.; SHEN, Z.; LIAN, C. Ectomycorrhizal fungal
communities associated with Masson pine (Pinus
massoniana) and white oak (Quercus fabri) in a
manganese mining region in Hunan Province, China.
Fungal Ecology, v. 9, p. 1-10, 2014.
https://doi.org/10.1016/j.funeco.2014.01.001
Silicon application and mycorrhiza inoculation promoted Leucaena leucocephala growth in a soil highly contaminated by manganese
Nativa, Sinop, v. 10, n. 3, p. 410-416, 2022.
416
IBRAM_Instituto Brasileiro de Mineração. Setor mineral 1°
trimestre 2020, 43. 2020. Acesso em: 16 de março de
2022. Disponível em: https://ibram.org.br/wp-
content/uploads/2021/02/PDF_DADOS_1oTRIM20
_16ABR20_FINAL-1.pdf
KABATA-PENDIAS, A. Trace elements in soils and
plants (4th ed.). Boca Raton: CRC Press, 2010. 548p.
KANG, X.; YU, X.; ZHANG, Y.; CUI, Y.; TU, W.; WANG,
Q.; LI, Y.; HU, L.; GU, Y.; ZHAO, K.; XIANG, Q.;
CHEN, Q.; MA, M.; ZOU, L.; ZHANG, X.; KANG, J.
Inoculation of Sinorhizobium saheli YH1 leads to
reduced metal uptake for Leucaena leucocephala grown
in mine tailings and metal-polluted soils. Frontiers in
Microbiology, v. 9, p. 1-13, 2018.
https://doi.10.3389/fmicb.2018.01853
KHAN, I.; AWAN, S. A.; RIZWAN, M.; ALI, S.; HASSAN,
M. J.; BRESTIC, M.; ZHANG, X.; HUANG, L. Effects
of silicon on heavy metal uptake at the soil-plant
interphase: A review. Ecotoxicology and
Environmental Safety, v. 222, p. 1-15, 2021.
https://doi.org/10.1016/j.ecoenv.2021.112510
LI, J.; JIA, Y.; DONG, R.; HUANG, R.; LIU, P.; LI, X.;
WANG, Z.; LIU, G.; CHEN, Z. Advances in the
mechanisms of plant tolerance to manganese toxicity.
International Journal of Molecular Sciences, v. 20, p.
1-15, 2019. https://doi.org/10.3390/ijms20205096
MOTAHARPOOR, Z.; TAHERI, H.; NADIAN, H.
Rhizophagus irregularis modulates cadmium uptake, metal
transporter, and chelator gene expression in Medicago
sativa. Mycorrhiza, v. 29, p. 389-395, 2019.
https://doi.org/10.1007/s00572-019-00900-7
PHILLIPS, J. M.; HAYMAN, D. S. Improved procedures for
clearing roots and staining parasitic and vesicular-
arbuscular mycorrhizal fungi for rapid assessment of
infection. Transactions of the British Mycological
Society, v. 55, p. 158-161, 1970.
https://doi.org/10.1016/s0007-1536(70)80110-3
SPAGNOLETTI, F.; CARMONA, M.; TOBAR, N. E.;
CHIOCCHIO, V.; LAVADO, R. S. Arbuscular
mycorrhiza reduces the negative effects of M. phaseolina
on soybean plants in arsenic-contaminated soils. Applied
Soil Ecology, v. 121, p. 41-47, 2017.
https://doi.org/10.1016/j.apsoil.2017.09.019
TANG, T.; TAO, F.; LI, W. Characterization of manganese
toxicity tolerance in Arabis paniculata. Plant Diversity,
v. 43, p. 163-172, 2020.
https://doi.org/10.1016/j.pld.2020.07.002
TEIXEIRA, P. C. T.; DONAGEMMA, G. K.; FONTANA,
A.; TEIXEIRA, W. G. Manual de Métodos de Análise
de Solo (3rd ed.). Brasília: Embrapa, 2017. 576p.
ULTRA, V. U.; MANYIWA, T. Influence of mycorrhiza and
fly ash on the survival, growth and heavy metal
accumulation in three Acacia species grown in Cu–Ni
mine soil. Environmental Geochemistry and Health,
v. 43, p. 1337-1353, 2021.
https://doi.org/10.1007/s10653-020-00627-x
UR RAHMAN, S.; XUEBIN, Q.; KAMRAN, M.; YASIN,
G.; CHENG, H.; REHIM, A.; RIAZ, L.; RIZWAN, M.;
ALI, S.; ALSAHLI, A. A.; ALYEMENI, M. N. Silicon
elevated cadmium tolerance in wheat (Triticum aestivum L.)
by endorsing nutrients uptake and antioxidative defense
mechanisms in the leaves. Plant Physiology and
Biochemistry, v. 166, p. 148-159, 2021.
https://doi.org/10.1016/j.plaphy.2021.05.038
VACULÍK, M.; KOVÁČ, J.; FIALOVÁ, I.; FIALA, R.;
JAŠKOVÁ, K.; LUXOVÁ, M. Multiple effects of silicon
on alleviation of nickel toxicity in young maize roots.
Journal of Hazardous Materials, v. 415, p. 1-13, 2021.
https://doi.org/10.1016/j.jhazmat.2021.125570
VIERHEILIG, H.; COUGHLAN, A. P.; WYSS, U.; PICHÉ,
Y. Ink and vinegar, a simple staining technique for
arbuscular-mycorrhizal fungi. Applied and
Environmental Microbiology, v. 64, p. 5004–5007,
1998. https://doi.org/10.1128/aem.64.12.5004-
5007.1998
VODNIK, D.; GRČMAN, H.; MAČEK, I.; VAN
ELTEREN, J. T.; KOVAČEVIČ, M. The contribution
of glomalin-related soil protein to Pb and Zn
sequestration in polluted soil. Science of the Total
Environment, v. 392, p. 130-136, 2008.
https://doi.org/10.1016/j.scitotenv.2007.11.016
WANG, B.; CHU, C.; WEI, H.; ZHANG, L.; AHMAD, Z.;
WU, S.; XIE, B. Ameliorative effects of silicon fertilizer
on soil bacterial community and pakchoi (Brassica chinensis
L.) grown on soil contaminated with multiple heavy
metals. Environmental Pollution, v. 267, p. 1-10, 2020.
https://doi.org/10.1016/j.envpol.2020.115411
YOST R. S.; FOX R. L. Influence of mycorrhizae on the
mineral contents of cowpea and soybean grown in an
oxisol. Agronomy Journal. v. 74, p. 475-481, 1982.
https://doi.org/10.2134/agronj1982.000219620074000
30018x
YOU, Y.; WANG, L.; JU, C.; WANG, G.; MA, F.; WANG,
Y.; YANG, D. Effects of arbuscular mycorrhizal fungi on
the growth and toxic element uptake of Phragmites australis
(Cav.) Trin. ex Steud under zinc/cadmium stress.
Ecotoxicology and Environmental Safety, v. 213, p. 1-
10, 2021. https://doi.org/10.1016/j.ecoenv.2021.112023
ZEHRA, A.; CHOUDHARY, S.; WANI, K. I.; NAEEM,
M.; KHAN, M. M. A.; AFTAB, T. Silicon-mediated
cellular resilience mechanisms against copper toxicity and
glandular trichomes protection for augmented
artemisinin biosynthesis in Artemisia annua. Industrial
Crops and Products, v. 155, p. 1-9, 2020.
https://doi.org/10.1016/j.indcrop.2020.112843
