Ecotoxicology and Environmental Safety 150 (2018) 76–85

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Cadmium induced changes in Solidago chilensis Meyen (Asteraceae) grown T

on organically fertilized soil with reference to mycorrhizae, metabolism,
anatomy and ultrastructure
Lidiane Silva Pereiraa, Romária Pereira de Araújob, Priscila Souza de Oliveirac,
Leandro Dias da Silvac, Patricia Alves Casaes Alvesa, Valéria Ferreira Fernandesd,
⁎
Eduardo Grosse,
a
Programa de Pós-Graduação em Biologia e Biotecnologia de Microorganismos, Universidade Estadual de Santa Cruz, Ilheus, Bahia, Brazil
b
Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Estadual de Santa Cruz, Ilheus, Bahia, Brazil
c
Programa de Pós-Graduação em Produção Vegetal, Universidade Estadual de Santa Cruz, Ilheus, Bahia, Brazil
d
Departmento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilheus, Bahia, Brazil
e
Departmento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz, Ilheus, Bahia, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Solidago chilensis Meyen (Asteraceae) is a medicinal important plant with few studies on nutrition and meta-
Medicinal plant bolism and none information on cadmium phytotoxicity. The objective of this study was to investigate Cd in-
Heavy metal duced responses on the growth and metabolism in S. chilensis and on arbuscular mycorrhiza (AM). The ex-
Manure periment was carried out in a greenhouse, consisting of a 5 × 4 factorial with five doses of manure (0, 3.5, 7, 14
Microorganism
and 21 g dm−3) and four doses of cadmium (0, 25, 50 and 75 mg dm−3) applied to a Dystrophic Ultisol. After
250 days of plant cultivation, biomass, nutrient content, photosynthetic rate, guaiacol peroxidase activity,
mycorrhizal colonization, glomalin content, anatomical and ultrastucture were evaluated. Plants were sig-
nificantly affected by interaction of manure and Cd doses with anatomical, ultrastructural, physiological and
nutritional modifications. Manure applied into Cd contaminated soil significantly improved mycorrhizal colo-
nization and glomalin production. The highest organic manure dose (21 g dm−3) alleviated toxicity symptoms of
Cd on S. chilensis.

1. Introduction
Solidago is the largest genus in Asteraceae, with species distributed
across different continents and recognized for their extensive ther-
apeutic use such as S. virgaurea L. in Asia and Europe, S. gigantea L., S.
odora Ait. and S. canadensis L. in North America and S. chilensis Meyen
in South America. The species S. chilensis is a natural medicinal plant
from Chile, distributed in South America, including the northeast,
center-west, southeast and south regions of Brazil (Russo and
Garbarino, 2008) and it has been widely studied in the substitution of
true arnica (Arnica montana L., Asteraceae) because it presents great
potential in the medical area as gastric protection, wound healing and
anti-inflammatory properties (Ramon Junior et al., 2015).
The Brazilian Ministry of Health has shown interest in stimulating
studies on the use of medicinal plants and medicinal products aiming at
the safe and effective use of pharmaceutical inputs of vegetal origin by
the population. In 2010, this Ministry published a list of 71 medicinal
plants evaluated with great potential to originate products of interest to
the Unified Health System (Portuguese abbreviation (SUS)), among
them S. chilensis, still with the synonym S. microglossa DC. Despite the
growing interest in medicinal species, it is also observed that natural
resources are being increasingly contaminated by heavy metals due to
chemicals disposal as well as untreated sewage, which can cause abiotic
and biotic changes at different levels on ecosystem. However, there are
few studies on the tolerance of Solidago genus to heavy metal, such as
cadmium (Cd), which is considered a natural occurring toxic element in
the environment at concentrations of 1 mg kg−1 (Sun et al., 2008). Due
to the application of contaminated agricultural inputs (in particular

Abbreviation: AMF, arbuscular mycorrhizal fungi; Cd, cadmium; SUS, unified health system; EEG, easily-extractable glomalin
⁎
Correspondence to: Departamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz, Rodovia Jorge Amado, km 16, Bairro Salobrinho, CEP 45662-900 Ilheus,
Bahia, Brazil.
E-mail addresses: lidiegas@hotmail.com (L.S. Pereira), roaraujo_bio@yahoo.com.br (R.P. de Araújo), priscilagronoma@gmail.com (P.S. de Oliveira),
leodias5@yahoo.com.br (L.D. da Silva), thyssa@gmail.com (P.A.C. Alves), val_bio1@hotmail.com (V.F. Fernandes), egross@uesc.br (E. Gross).

https://doi.org/10.1016/j.ecoenv.2017.12.017
Received 17 August 2017; Received in revised form 1 December 2017; Accepted 9 December 2017
Available online 19 December 2017
0147-6513/ © 2017 Elsevier Inc. All rights reserved.
L.S. Pereira et al.
pesticides and phosphate manures), the generation of untreated was-
tewater and industrial waste Cd can reach concentrations up to
1020 mg kg −1 in the environment (Brown et al., 1994).
Cd presents no evidence of essentiality to humans and is toxic even
at extremely low levels, being a potent cancer-causing agent, accumu-
lating especially in the kidney, causing gastric and intestinal dysfunc-
tions, hypertension, anemia and pulmonary emphysema and, moreover,
is more easily absorbed by plants compared to other heavy metals
(Burke et al., 2016). When in high concentrations and for prolonged
periods of exposure, this element has a toxic effect, inactivating en-
zymes and causing human cells death (Templeton and Liu, 2010). The
potential toxicity of Cd to soil microbiota and plants is directly related
to the availability of this element in solution, which is affected by both
biotic and abiotic factors, and the soil microorganisms themselves can
influence the mobility and bioavailability of Cd in the soil-plant system
(Pelfrêne et al., 2012).
S. chilensis presents symbiotic association with arbuscular mycor-
rhizal fungi (AMFs), which are important components of the soil mi-
crobiota and that promote benefits for the growth of plants in soils
contaminated by heavy metals (Hassan et al., 2013). In these soils, the
AMFs can contribute by forming a "barrier" for the metal ions, in-
creasing the tolerance of the plant, accumulating the heavy metal in the
fungal structures, reducing the translocation of the metal to the plant,
retaining in the soil, changing the bioavailability of the toxic element,
and increasing phytoextraction capacity (Cicatelli et al., 2014).
In addition to AMF, organic fertilizers were tested to reduce heavy
metal effects on contaminated soil and as source of nutrients to the
plants (Pardo et al., 2011; Park et al., 2011). Manures and organic
fertilizers have been used to crop production improving soil fertility
and stimulating microbial activity (Jannoura et al., 2014).
Although some studies have focused on the importance of AMF and
manures in reducing Cd availability, no study was conducted to eval-
uate the effects of using manure to alleviate Cd toxicity in the growth,
nutrition, anatomy and metabolism of Solidago chilensis mycorrhizal
plants. Therefore, the objective of the present study was to evaluate the
tolerance of this species of medicinal plants to Cd, testing the hypoth-
esis that doses of manure applied to the soil can reduce the availability
and absorption of this toxic heavy metal also contributing to the my-
corrhizal colonization increase.
2. Material and methods
2.1. Experimental conditions
The experiment was evaluated in a greenhouse at the State
University of Santa Cruz (UESC), Ilhéus, Bahia, Brazil (14° 47 ′S, 39° 10′
W) for a period of 250 days. The S. chilensis seedlings were produced
from plant cuttings grown in UESC Medicinal Plants in plastic cups with
uncontaminated soil for 60 days and then a seedling was transplanted
for each plastic pot containing 5 kg of soil (typical dystrophic Ultisol)
collected from the surface layer (0–20 cm). This soil presented a sandy
texture, containing 74.06% of sand, 12.61% of silt and 13.33% of clay.
The soil chemical characterization was performed according to
Embrapa (2011). Soil pH was determined at a soil: water ratio of 1:2.5
(m/m). Magnesium (Mg), calcium (Ca), aluminum (Al) were extracted
with 1 M KCl and quantified through atomic absorption spectrometry
(for Ca and Mg) and titration with NaOH 0.025 M (for Al). Potassium
(K) and phosphorus (P) were extracted using Mehlich 1 method and
quantified through flame spectrometry (K) and blue-Mo method (P).
Soil organic matter (SOM) was determined using the Walkley and Black
procedure. Sulphur (S-SO4) was extracted with 1 M HCl and precipitate
with BaCl2. The micronutrients copper (Cu), iron (Fe), zinc (Zn) and
manganese (Mn) were extracted by Mehlich 1 and quantified by atomic
absorption spectrometry, and boron (B) was extracted with hot water
and determined by colorimetric by azometine-H method. Cation ex-
change capacity (CEC) was estimated through the sum of Ca, Mg, K, and
77
Ecotoxicology and Environmental Safety 150 (2018) 76–85
Al. The soil presented the following results: pH = 4.7; OM (organic
matter) = 2.6%; exchangeable cations (cmolc dm−3): K = 0.09; Ca =
0.6; Mg = 0.3;Al = 1.2; H +Al = 8.4; sum of bases = 0,99; ECEC =
2.19; P = 7 mg dm−3; S = 5 mg dm−3; micronutrients (mg dm−3): Cu
= 0.8; B = 0.6; Mn = 46; Fe = 183; Zn = 1.6; Cd = not detected.
The experimental design was completely randomized and consisted
of 120 sample units divided into 20 treatments. The experiment con-
sisted of a 5 × 4 factorial, with five doses of the manure (0, 3.5, 7, 14,
21 g dm−3), four doses of cadmium (0, 25, 50 and 75 mg dm−3 applied
as CdCl2), with six replicates (sample units consisting of one plant per
pot). The analysis of the manure (obtained from manure and urine
cattle) was carried out in the Laboratory of Organic Matter and
Residues of the Department of Soils of the Federal University of Viçosa,
Viçosa - MG, and resulted in the following values for micronutrients (in
mg kg−1) Cu = 20.27; Fe = 2564.475; Mn = 214.6; Zn = 119.75; Cd
= Not detected; Pb = 6.6; Cr = 30.77; Ni = 14.8; and for macro-
nutrients (in %): Ca = 2.06; Mg = 0.15; K = 0.21; Na = 3.61; P =
0.05; Total Organic Carbon = 40.26. The humidity of the pots was
maintained at 70% of the field capacity, which was previously estab-
lished.
2.2. Harvesting and analysis of plants
At the end of the experiment (seven months of plant growth) the
plants were harvested in root system, stem and leaves to determine the
dry biomass, after being kept in an oven with forced air circulation at
60 °C until constant weight, followed by weighing in semi-analytical
scale. The roots, stems and branches and leaves were ground in a Wiley
type mill, placed in properly identified paper bags, which were sent to
the Laboratory of Soil Analysis, Plant Tissue and Fertilizer of the Soils
Department of the Federal University of Viçosa, city of Viçosa (MG), for
their chemical analyses to be performed. The methodologies for these
analyzes were: P: colorimetry (metavanadate ammonium method); S:
colorimetry (turbidimetry of barium sulfate); K, Ca, Mg, Cu, Fe, Mn and
Zn: atomic absorption spectrophotometry; Sulfuric digestion for N-total;
B: colorimetry (Azomethine H method).
2.3. Quantification of mycorrhizal colonization
In order to estimate the percentage of mycorrhizal colonization in
the samples of the roots of Solidago chilensis Meyen, the bleaching
technique was used with 10% KOH and stained with blue pen in ac-
cordance with the methodology described by Phillips and Hayman
(1970) modified followed by the technique used by Mcgonigle et al.
(1990).
2.4. Extraction and quantification of spores
The spores of the rhizospheric soil samples were extracted following
the technique of decantation and wet sieving combined with the tech-
nique of centrifugation in 50% sucrose solution (Gerdemann and
Nicolson, 1963; Jenkins, 1964).
2.5. AMF identification
The previously isolated spores were separated into groups (mor-
photypes) under stereomicroscope and then mounted on permanent
slides with PVLG resin and Melzer reagent (Morton et al., 1993). These
slides were then placed in an oven at temperatures of 40 °C for ap-
proximately 30 h to accelerate the drying of the resin, favoring spores
breaking from light pressures with a rubber pencil in circular move-
ments on the cover plate against the slide. The spores preserved in
slides were observed under optical microscope (increase of 1000×)
highlighting the morphological characteristics, such as size (in μm),
shape, coloration, structure and ornamentation of walls, type of spore
hyphae and germination mode.
L.S. Pereira et al.
The identification was made following the manual by Schenck and
Pérez (1988), descriptions provided by the INVAM database (The In-
ternational Culture Collection of Arbuscular and Arbuscular Mycor-
rhizal Fungi), available on the Internet at: http://fungi.invam.wvu.edu/
the-fungi/species-descriptions.html, and related works.
2.6. Content of easily-extractable glomalin
The estimation of the easily-extractable glomalin content (EEG) for
the soil was performed according to the Wright and Upadhyaya (1996)
methodology and quantified by means of the Bradford's (1976) micro-
titer technique.
2.7. Guaiacol peroxidase
The activity of the guaiacol peroxidases was determined according
to methodologies proposed by Pirovani et al. (2008). We have used
200 mg of leaf sample macerated in liquid nitrogen, adding a phosphate
buffer (50 mmol L−1, pH 6.0), with ultrasonication (8 pulses of 10 s
each, amplitude of 70–80%, with 10 s interval) in an ultrasonic pro-
cessor (Ultrasonic processor Gex 130, 130 W) on ice until tissue rup-
ture. Next, the samples were centrifuged for 10 min at 15,000 rpm. We
have used microplates with 96 apertures with capacity for 300 μl,
containing 140 μl of guaiacol 2 × 40 mmol L−1 peroxidase buffer of
guaiacol, 0.06% H2O2 and sodium phosphate (20 mmol L−1, pH 6.0),
139 μl of phosphate buffer (50 mmol L−1, pH 6.0) and 1 μl of enzyme
extract, previously diluted. The absorbance change at 470 µm was
monitored for 60 s of reaction at 25 °C in a microplate reader spectro-
photometer (VERSAmax). The activity of peroxidases was expressed
with increasing guaiacol consumption in μmol s−1 g −1 of lyophilized
biomass.
2.8. Leaf gas exchange and chlorophyll
The measurements of gas exchange were performed on mature
leaves of the stem apex from the last release, at 250 days, in which the
liquid photosynthetic rate, the transpiratory rate and the stomatal
conductance were measured using a portable Li-6400 photosynthetic
measurement system (IRGA LI-6400, LICOR®, Nebraska/USA). During
the measurements, the photo synthetically active radiation (RFA) and
the atmospheric CO2 concentration were kept constant under 1000
μmol photons m−2 s−1 and approximately ± 380 μmol mol−1.
2.9. Anatomical features
We have selected fully mature leaves, and the collections were al-
ways done in the middle region of the leaf. Three replications of each
treatment were collected for a total of 45 samples, each replicate cor-
responding to one treatment plant. The selected samples were fixed in
2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2, dehy-
drated in a ethanol series and embedded in methacrylate resin
(HistoresinLeica; Leica Microsystems, Heidelberg, Germany). Cross
Section (5 µm thick) were obtained using a rotary microtome (Leica
RM2265; Deerfield, IL), stained with toluidine blue (pH 4.7) (O’Brien
et al., 1964). Analyses and documentation of the results were carried
out under a Leica® photon microscope model DMI 3000 B. In these
analyses, the measurements were made in 10 different fields of each
sample, the thickness of the epidermis in the adaxial and abaxial sides
the palisade and spongy parenchyma using the Leica application suite
V3 software.
2.10. Ultrastructural analysis of the leaf
For the ultrastructural analysis, the collected and selected materials
were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at
pH 7.2, rinsed in the same buffer (four times, 10 min each), postfixed in
78
Ecotoxicology and Environmental Safety 150 (2018) 76–85
1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 h and
rinsed in the same buffer (four times, 10 min each). After dehydration
in an ethanol series, samples were embedded in LR-white resin. Sections
of 70–80 nm thickness were obtained using an ultramicrotome (Leica
EMUC 6; Vienna, Austria). These sections were collected on copper
grids and contrasted with 5% uranyl acetate aqueous solution (5 min)
and lead citrate (3 min). Observations and images were obtained using
a Morgagni transmission electron microscope FEI 268D at 80 kV of
acceleration voltage.
2.11. Statistical analysis
Data were submitted to factorial ANOVA, followed by a comparison
test of means (Tukey) with probability level at 5%.
3. Results
3.1. Plant biomass
Plants of S. chilensis presented significant differences (p≤0.05) in
the biomass production influenced by the interaction between Cd and
the manure doses applied to the Ultisol (Fig. 1). The application of Cd in
the soil at doses of 25 and 50 mg dm−3 resulted in a decrease in leaf,
stem and root S. chilensis production when compared to the un-
contaminated treatment (Fig. 1). Both leaves and stems showed greater
biomass production when the highest dose of manure (21 g dm−3) was
applied in the treatment contaminated with 25 mg of Cd (Fig. 1A and
B). The biomass production of the root system was lower in plants
grown in soil contaminated with 50 mg dm−3 of cadmium and ferti-
lized with low doses of the manure (0, 3.5, 7 and 14 g dm−3) differing
statistically from those grown in fertilized soil with the highest dose of
the manure (21 g dm−3) even in the soil contaminated with the highest
dose of cadmium (Fig. 1C). Total biomass plant production was pre-
judiced by highest dose of Cd but the highest dose of manure applied in
soil alleviates the negative influence of this metal on plant growth
(Fig. 1D).
Data from plants grown in soil contaminated with the highest dose
of Cd (75 mg dm−3) could not be included in the statistical analyses
due to the low survival rate, and the few plants that survived (nine in
total) did not develop and produced insufficient biomass for the ana-
lyses.
3.2. Mycorrhizal colonization and glomalin production
The average values of mycorrhizal colonization in the roots of S.
chilensis were significantly influenced by the interaction between the
doses of the manure and of Cd applied to the Ultisol. The treatments
that received 50 mg dm−3 of Cd in the soil increasing root colonization
by AMF at all doses of manure tested, differing statistically from
treatments with soil non-contaminated by Cd (Fig. 2A).
Glomalin levels did not show significant differences in the non-
contaminated heavy metal soil across all doses of the manure tested
(Fig. 2B). However, as a result of the interaction between the manure
and Cd added to the soil, at 25 mg dm−3 of Cd there were significant
differences in the average values of easily-extractable glomalin from the
soil between doses 0 and 3.5 g dm−3 and 7, 14 and 21 g dm−3 of the
manure (Fig. 2B). At the dose of 50 mg dm−3 of Cd applied to the soil
there were no significant differences between the doses of manure but
the average values of glomalin differed significantly from those ob-
served for uncontaminated soil (Fig. 2B).
It was also observed that the number and diversity of fungal
structures increased as the soil Cd concentrations were elevated. The
number of structures (hyphae, arbuscules, spores and vesicles) differed
significantly between treatments with uncontaminated and con-
taminated soil, and a higher number of these structures was observed in
the roots of the plants grown in soil contaminated with 25 and
L.S. Pereira et al.
Fig. 1. (A) Mean values of leaf biomass, (B) stem biomass, (C) root biomass and (D) total biomass of Solidago chilensis plants submitted to cadmium (0, 25 and 50 mg dm−3) and manure
(0, 3.5, 7, 14 and 21 g dm−3) doses applied to typical dystrophic Ultisol. Same upper case letters (among Cd in the same dose of manure) and lowercase (among manure in the same dose
of cadmium) do not differ significantly from each other by the Tukey test (p ≤ 0,05).
Fig. 2. (A) Mean values for mycorrhizal colonization of Solidago chilensis roots submitted
to cadmium 0, 25 and 50 mg dm−3) and manure (0, 3.5, 7, 14 and 21 g dm−3) doses
applied to a typical dystrophic Ultisol. (B). Mean values for glomalin extracted from this
soil after cultivation with S. chilensis.
79
Ecotoxicology and Environmental Safety 150 (2018) 76–85
50 mg dm−3 of Cd and the highest dose of the manure applied
(Supplementary Fig. S1).
3.3. Taxonomy of the AMF
Among the spores of AMF extracted and isolated from soil where S.
chilensis plants grown it was possible to morphologically identify eight
genera and species of fungi (Supplementary Table S1). They were dis-
tributed in the rhizospheres of the plants from treatments with addition
of Cd and manure. It was observed that the species Dentiscutata het-
erogama and Scutellospora sp. were found in larger quantity of treat-
ments, Glomus macrocarpum including those containing Glomus micro-
aggregatum, was found only in the treatments with Cd and without
addition of manure, and the species of the genus Racocetra were found
only in the control treatment (Supplementary Table S1).
3.4. Cadmium in the soil
In terms of the available cadmium content in the soil, significant
interaction was observed for Cd and the manure doses applied to the
Ultisol (Fig. 3). As expected, there was a significant difference between
the uncontaminated soils and those in which 25 and 50 mg dm−3 of
cadmium were applied, and in the soil in which 50 and 25 mg dm−3 of
Cd was added at the dose of 21 g dm−3 of manure, there was a re-
duction of the available Cd content, being significant in soil con-
taminated with 50 mg dm−3 of cadmium as observed in Fig. 3.
3.5. Cadmium in the plant
The total cadmium content in the S. chilensis plants presented sig-
nificant differences (p ≤ 0.05) and were influenced by the interaction
between the Cd and the doses of manure applied to the soil (Fig. 4). As
expected on uncontaminated soil, the different doses of manure did not
present significant differences in the contents of Cd in all parts of the
plant (Fig. 4A, B and C) since both, soil and manure not presented Cd in
composition. For soil treatments in which 25 and 50 mg dm−3 of Cd
L.S. Pereira et al.
Fig. 3. Concentrations of cadmium in the typical dystrophic Ultisol submitted to Cd (0, 25
and 50 mg dm−3) and manure (0, 3.5, 7, 14 and 21 g dm−3) doses after S. chilensis cul-
tivation. Lowercase letters (between doses of manure at the same dose of cadmium) and
capital letters (between doses of Cd at the same dose of manure) do not differ significantly
from each other by the Tukey test p ≤ 0.05.
were applied with the highest dose of manure (21 g dm−3) it was
possible to demonstrate a reduction in the cadmium concentration in
the leaves, stems and roots of S. chilensis (Fig. 4A, B, C). It was also
possible to observe that the part of the plant that presented the highest
content of this metal was the root (Fig. 4C). Apparently the two inter-
mediated doses of manure (7 and 14 g dm−3) stimulated plant growth
and consequently promotes roots uptake of Cd available in the soil.
However manure applied in the highest dose in soil (21 g dm−3) acts
reducing the Cd availability consequently reducing in plant tissues
(Fig. 4D).
3.6. Plant nutrient content
The nutrient content averages found in the dry biomass samples of
the S. chilensis leaves presented wide variability for all analyzed macro
and micronutrients. No clear tendency of reduction or increase in the
nutrient content was observed as the doses of cadmium or manure
Fig. 4. Cadmium concentrations in the leaf (A), stem (B), root (C) and total (D) on Solidago chilensis plants submitted to 0, 25 and 50 mg dm−3 of Cd 0, 3.5, 7, 14 and 21 g dm−3 of
manure. Lower case letters (between doses of manure in the same dose of cadmium) and upper case letters (between doses of Cd in the same dose of the manure) do not differ significantly
from each other by the Tukey test p ≤ 0.05.
80
Ecotoxicology and Environmental Safety 150 (2018) 76–85
applied to the soil increased (Table 1). There was therefore no sig-
nificant interaction between the cadmium and manure doses on these
nutrient contents in the S. chilensis leaves with the exception of Zn and
Mn contents that at the doses of 25 and 50 mg dm−3 of cadmium ap-
plied, increased and reduced, respectively, when compared to the soil
without addition of cadmium (Table 1).
In regards to the nutrient contents of the stem, plants grown in soil
contaminated with 25 mg dm−3 of Cd showed a tendency to increase
the N, P, K and S macronutrients in relation to those of uncontaminated
soil, and a decrease in Fe, Zn and Mn in relation to those grown in soil
not contaminated by Cd, described in Table S2 (Supplementary mate-
rial).
For the macro and micronutrient contents in the root samples, an
increase in N levels of plants cultivated in soil contaminated with Cd
was observed in relation to the plants grown in uncontaminated soil
(Supplementary Table S3). In regards to the micronutrients, there was a
tendency for a decrease in Cu and Zn contents found in the root tissues
of plants cultivated in soil contaminated with Cd in comparison to those
in plants grown in uncontaminated soil, described in Table S3
(Supplementary material).
3.7. Guaiacol peroxidase
The activity of guaiacol peroxidase (POD) in leaf and roots of S.
chilensis presented a significant difference (p ≤ 0.05), and seem to be
influenced by the interaction between Cd and manure doses applied
into the soil. A higher activity of these peroxidases was observed in the
leaves than in the S. chilensis root when Cd was applied to the soil. We
have also observed a higher activity of guaiacol peroxidases at the dose
of 25 mg dm−3 of Cd applied to the soil concomitantly with doses of 3.5
and 7 g of manure, as observed in (Supplementary Fig. S2).
3.8. Gas exchange
There was a tendency of decrease in the net photosynthetic rate,
L.S. Pereira et al. Ecotoxicology and Environmental Safety 150 (2018) 76–85

Table 1
Mean values of macro and micronutrient contents in leaves of Solidago chilensis submitted to different doses of cadmium and manure.

Macronutrients (%) Micronutrients (mg kg−1)

Cd (mg dm−3) Manure (g dm−3) N P K Ca Mg S Cu Fe Zn Mn B

0 0 2.03 0.22 1.24 1.65 0.48 0.12 6.96 305.18 66.19 306.20 111.67
3.5 1.79 0.24 1.63 1.82 0.50 0.12 8.25 298.71 85.79 389.69 127.58
7 2.15 0.26 1.62 1.86 0.56 0.11 8.25 223.79 78.03 341.03 102.19
14 1.63 0.30 1.42 2.00 0.54 0.11 8.70 240.45 78.59 402.31 109.84
21 1.91 0.28 1.59 2.10 0.55 0.12 9.14 222.29 84.54 409.03 108.72
25 0 2.17 0.29 2.01 1.71 0.56 0.16 6.48 317.98 121.83 165.46 126.37
3.5 2.33 0.31 1.86 2.05 0.61 0.17 7.08 311.27 110.53 228.60 93.41
7 2.38 0.35 2.19 1.93 0.63 0.19 7.71 178.76 113.08 158.43 97.40
14 2.65 0.32 2.15 1.86 0.58 0.18 7.30 153.94 185.50 217.64 124.97
21 2.47 0.33 1.76 1.68 0.61 0.18 6.43 201.30 110.64 170.21 95.94
50 0 2.65 0.32 1.46 2.09 0.60 0.18 5.89 292.39 126.05 293.90 99.43
3.5 2.47 0.30 1.59 1.83 0.57 0.17 6.06 211.51 124.64 243.83 130.21
7 2.71 0.29 1.23 2.24 0.56 0.18 6.62 216.72 150.07 258.70 130.42
14 2.84 0.35 1.60 1.70 0.54 0.17 6.73 181.25 131.50 212.79 116.29
21 2.43 0.33 1.71 2.22 0.62 0.14 7.10 165.18 112.15 333.80 105.20

stomatal conductance to water vapor and transpiratory rate in plants
grown in soil contaminated with 50 mg dm−3 of Cd. This tendency was
also observed for plants grown in soil contaminated with 25 mg dm−3
of Cd. The photosynthetic rate, stomatal conductance, and transpiratory
rate of plants cultivated in the uncontaminated soil decreased at the
dose of manure 14 g dm−3 applied to the soil, rising at the dose
21 g dm−3 (Supplementary Fig. S3).
3.9. Plant anatomy
The leaves of S. chilensis presented non-stratified uniseriate epi-
dermis composed by cells of rectangular shape, covered by a thick cu-
ticle with dorsiventral mesophyll, with two to three layers of short
palisade parenchyma cells in both upper and lower epidermis and one
to two central layers of colorless cells (Fig. 5A and B). We observed a
disorganization of the palisade parenchyma cells and rupture of the
epidermis in leaves collected from plants grown with heavy metal (25
and 50 mg dm−3 of Cd) and without manure addition (Fig. 5C and E).
Anatomy of the leaves obtained from plants grown on soil with highest
Cd dose demonstrated tissue disorganization, with changes at shape and
arrangement of palisade parenchyma cells probably due to the increase
of intercellular spaces (Fig. 5E). On the other hand, plants submitted to
21 g dm−3 of manure and without heavy metal exposure (Fig. 5B)
presented similar tissue organization to the control plants (Fig. 5A).
Plants grown in soil contaminated with 25 and 50 mg dm−3 of Cd and
fertilized with 21 g dm−3 of manure presented leaf anatomy better
preserved and tissue organization without intercellular spaces (Fig. 5D
and F, respectively) when compared to those submitted to different Cd
doses without manure application (Fig. 5C and E).
The thickness of the cells on palisade parenchyma from adaxial and
abaxial leaf surfaces, except for the plants submitted to the highest dose
of Cd and manure (50 mg dm−3 and 21 g dm−3, respectively) was re-
duced in all other treatments in comparison to the control
(Supplementary Fig. S4A).
The colorless cells observed between the layers of palisade par-
enchyma cells underwent a significant reduction in their thickness on
plants grown with 25 and 50 mg dm−3 of Cd and without manure
fertilization differing from the thickness of the cells of control plants
and from those with the highest dose of manure applied in soil
(Supplementary Fig. S4B).
3.10. Plant ultrastructure
S. chilensis leaf ultrastructural study demonstrated changes on par-
enchymal cells sampled from the plants submitted to 25 and
50 mg dm−3 of Cd and without manure fertilization (Fig. 6C and E,
respectively) when compared with mesophyll cells structure of leaves
sampled from control (Fig. 6A) and manure fertilized (Fig. 6B, D and F)
plants. Main changes observed were a higher frequency of plastoglobuli
and starch grains in addition to dilated chloroplasts (Fig. 6C and E). It
was also verified that, when submitted to 50 mg dm−3 of Cd and
without the manure, the chloroplast presented disorganization in the
thylakoids, and fragments of chloroplasts were lose in the cytoplasm in
addition of starch grains production that has also been evidenced
(Fig. 6E).
Leaves samples from fertilized plants (21 g dm−3 of manure) pre-
sented chloroplasts of mesophyll cells with apparently organization and
integrity of (Fig. 6B, D and F). The presence of grana and the decreased
of starch grains resembling the ultrastructure of control cells leaves
(Fig. 6A). It was still possible to observe that in cells of plants cultivated
without Cd and with 21 g dm−3 manure fertilization there was a pre-
sence of fewer plastoglobuli (Fig. 6B, D and F).
4. Discussion
The biomass production of S. chilensis in treatments with soil con-
taminated with Cd was favored with the application of the maximum
dose of manure, which may have improved soil structure, availability of
nutrients and complex heavy metals as suggested by (Oldare et al.,
2011). The application of manures is a common practice worldwide to
fertilize the soil and consequently increase crop production (Ahmad
et al., 2015). Cd is one of the heavy metals that most negatively affect
plant growth and the application of manure can not only improve plant
nutrition but also reduce the availability of heavy metals in the soil as
seen in our experiment with S. chilensis grown in dystrophic Ultisol.
Another important influence of the application of manure is the
stimulation of the microbial activity, as demonstrated by the results of
mycorrhizal colonization of the roots of S. chilensis in the highest doses
of manure applied to the soil. AMF are microorganisms that may exert a
protective role for plants against heavy metal toxicity such as from Cd,
and many plants have shown greater tolerance to heavy metals when
associated with mycorrhizal fungi (Yang et al., 2015; Doubkorá and
Sudová, 2016). The high concentration of Cd did not have a negative
effect on the mycorrhizal colonization of S. chilensis roots. On the
contrary, they increased their percentage of colonization as well as the
propagation of their fungal structures, possibly minimizing the dele-
terious effects of Cd on the plant by the absorption of this metal (Hy-
pothesis 1). In addition, the hypothesis that hyphal cells have been
found to be involved in fungal cells has been shown to be associated
with organic substances (Bedini et al., 2009). In agreement with the
results observed for S. chilensis, other studies have pointed to the ab-
sence of detrimental effect of Cd on AMF colonization and several

81
L.S. Pereira et al.
factors such as plant species and AMF, contamination content and soil
characteristics may influence the tolerance of mycorrhiza to this metal
(Rivera-Becerril et al., 2002; De Andrade et al., 2008; Saad et al., 2013;
Garg and Bhandari, 2014).
High concentrations of heavy metals may exert a selection on AMF
populations, favoring the dominance of more adapted species, pro-
viding different effects on plants according to the fungal species
(Schneider et al., 2013). Metal-tolerant AMF may play an important
role in phytoremediation and some studies have demonstrated the de-
leterious effects of metals on microbial diversity and microbial activity
in the soil (Orowska et al., 2011; Glassman and Casper, 2012). Among
the species of AMF identified in our study, Dentiscutata heterogama was
found in treatments with doses of Cd and without addition of manure
(25 and 50 mg dm−3) as well as in treatment with 25 mg dm−3 and
21 g dm−3 of manure and it has already been tested in other studies
with heavy metals promoting, in certain cases, higher contents of car-
bohydrate and larger size in terms of height of the host plant
(Ambrosini et al., 2015). The Glomus genus found in the treatment with
25 mg dm−3 of Cd without manure has also been reported in several
works due to its capacity of tolerance in contaminated environments as
other species of Scutellospora (Zaefarian et al., 2010; Carrasco et al.,
2011; Chanda et al., 2014; Leal et al., 2016).
Symbiotic efficiency of the AMFs varies between fungal species and
host plants, and this may be more evident in situations of stress, such as
contamination of soil by heavy metals. The protective effect of these
fungi also depends on the chemical concentrations and forms of soil
contaminants (Ambrosini et al., 2015).
AMF produce a protein called glomalin, which plays a key role in
soil aggregation and biostabilization, and which binds to pollutants and
82
Ecotoxicology and Environmental Safety 150 (2018) 76–85
Fig. 5. Sections of Solidago chilensis leaves collected
from plants submitted to different doses of cadmium
(0, 25 and 50 mg dm−3) and manure (0 and
21 g dm−3). (A) control treatment leaf (from plant
grown on soil without Cd and manure addition)
showing the tissue distribution; (B) leaf section from
plant fertilized with 21 g dm−3 of manure and
without Cd application in soil showing similar or-
ganization of tissue to the control leaf plant; (C)
space between cells of the palisade parenchyma
(arrowhead) and epidermal rupture (arrow) in leaf of
plant submitted to 25 mg dm−3 of Cd and without of
manure addition; (D) leaf from plant submitted to
25 mg dm−3 of Cd and fertilized with 21 g dm−3 of
manure showing tissue organization without inter-
cellular spaces; (E) large intercellular spaces and
epidermal disruption observed in leaf sampled from
plant grown at 50 mg dm−3 of Cd and without
manure additions in soil; (F) sample of leaf from
plant submitted to 50 mg dm−3 of Cd and fertilized
with 21 g dm−3 of manure when we can observed a
typical tissue organization of S. chilensis leaves.
metal ions present in the soil (Bedini et al., 2009). The high content of
easily-extractable glomalin in treatments with soil contaminated with
both 25 and 50 mg dm−3 of Cd at all doses of manure applied can be
explained by the increase of mycorrhizal colonization in the plants of
these treatments. The action of glomalin on the unavailability of Cd in
the soil may have directly or indirectly contributed to the increase of
biomass in the plants, provided that this protein can be attached to
cations in amounts that vary in different soils and because it has high
binding capacity to certain heavy metals (Cu, Pb, and Cd), which is
important for the biostabilization of contaminated soils (González-
Chávez et al., 2004; Chen et al., 2005).
The toxicity of Cd depends on exposure time, metal concentration,
and soil chemical and physical characteristics such as organic matter
content, cation exchange and pH (He et al., 2015). S. chilensis presented
the highest concentration and accumulation of Cd in the root system,
which is a behavior similar to that observed for several other plant
species, such as Bidens pilosa (Sun et al., 2008) and Salix spp. (Iori et al.,
2014). According to Verbruggen et al. (2009), a higher concentration of
metals in the root is characteristic of intolerant plants, while the ability
to metals translocation to the shoot is recognized as a tolerance factor.
The increased concentration of Cd in the roots is not only due to the
increase in the absorption of this element, but due to the concomitant
decrease of the dry matter production. In S. chilensis, although there
was a high concentration of Cd in the roots, this element was also found
in leaves and stems, indicating that it is not totally immobilized in the
root system. This result is similar as also observed to Zea mays (Dresler
et al., 2014) and Theobroma cacao (de Araújo et al., 2017).
At the end of the experiment we were able to verify that the highest
dose of manure reduced the amount of available Cd in typical
L.S. Pereira et al. Ecotoxicology and Environmental Safety 150 (2018) 76–85

Fig. 6. Electromicrography of mesophyll cells from S. chilensis plants submitted to different doses of cadmium (0, 25 and 50 mg dm−3) and manure (0 and 21 g dm−3). (A) A healthy
ultrastructural aspect of chloroplasts from a control plant leaf. In the right corner: a detail of thylakoid organization. (B) Chloroplast with well organized thylakoids and few plastoglobuli
(arrow) from a plant grown on soil without Cd and with 21 g dm−3 of manure fertilization. (C) Chloroplast aspect from a plant submitted to 25 mg dm−3 of Cd and without manure
addition, evidencing changes and presence of starch grains and plastoglbuli (arrow). (D) A well preserved chloroplast from a plant submitted to 25 mg dm−3 of Cd and 21 g dm−3 of
manure, presenting grana. (E) Chloroplast structure from a 50 mg dm−3 of Cd and without manure fertilization plant, evidencing changes on thylakoids organization and a great number
of starch grains. In the right corner: detail showing a part of chloroplast (arrow). (F) Mesophyll cell from a plant submitted to 50 mg dm−3 of Cd and 21 g dm−3 of manure showing well
preserved chloroplast presenting integral grana. Starch (S), thylakoid (t), mitochondria (m), thylakoids grana (gt). Scale bars: A and D = 0.5 µm; B, C, E and F = 1 µm.

dystrophic Ultisol, indicating the complexation with the molecules,
probably with humic and fulvic acids present in organic compounds of
manure, as suggested by Zhang et al. (2015) and Moreno-Jiménez et al.
(2016). Manure has been used to improve soil fertility and to increase
crop yield, not only because of its nutritional value but also because the
capacity for cation exchange (Kiran et al., 2017). In addition, humic
acid present in organic composts can form complexes with heavy me-
tals, altering soil pH and reducing heavy metal bioavailability in soil
(Zhou et al., 2015; Khan et al., 2017).
Our study demonstrated a high concentration of cadmium in S.
chilensis plants grown on soil with 50 mg dm−3 of Cd and fertilized with
7 and 14 g of dm−3 of manure, which probably stimulated the plant
growth and consequently promotes root uptake of available Cd. In
addition, it is also possible that there is a synergism between nutritional
elements (Li et al., 2016; Mwamba et al., 2016; Rizwan et al., 2017). It
is important to notice that in the highest dose of cattle manure fertili-
zation (21 g dm−3) it acts reducing the Cd availability in soil and
consequently reducing in S. chilensis tissues, probably chelating this
toxic element with some organic constituent now present in high con-
tent due the quantity applied in typical dystrophic Ultisol. This is a
valuable information that we obtained from our experiment showing
that the correct dose of manure is fundamental to reduce the Cd content
in plant and the soluble/exchangeable Cd in soil.
The excess heavy metals generally stimulate the formation of re-
active oxygen species (ROS), which are controlled by various me-
chanisms, such as glutathione, ascorbic acid and phenolic metabolites
(Babula et al., 2015). The increase of Cd content in the plant tissues of
S. chilensis with the concomitant increasing of guaiacol peroxidase ac-
tivity and reduction of the photosynthetic rate indicate the stress caused
by the doses of Cd applied into the soil. A study with L. satium exposed
to Cd indicated that free radical formation resulted in a significant in-
crease in lipid peroxidation and membrane rupture (Gill et al., 2012).
On plants with the highest dose of Cd (50 mg dm−3) and manure
(21 g dm−3) the decreasing of photosynthetic rate can be explained by
the oxidative stress that Cd can cause, thus damaging proteins and
membrane whereas the elevation of stomatal conductance and tran-
spiration rate in the same plants suggests that the manure facilitated
stomatal opening and CO2 entry (Ashfaque et al., 2017).
Due to the translocation of the heavy metals to the shoot, especially
to the leaves, the plant can undergo morphological modifications. Cd is
a toxic pollutant that causes anatomical and structural changes in the
roots and leaves of plants (Najeeb et al., 2011) and according to
Kovácik and Babula (2017) plants submitted to Cd presented cell wall
of the vessels and the adjacent parenchyma with anatomical alterations.
Our study showed that plants of S. chilensis, when submitted to 25 and
50 mg dm−3 Cd doses without manure may suffer severe anatomical
changes in the leaves especially in the thickness and spatial organiza-
tion of mesophyll tissues. These changes were also described for soy-
bean, Brachiaria decumbens and tomato leaves when plants were sub-
mitted to doses of heavy metals (Weryszko-Chmielewska and Chwil,
2005; Gomes et al., 2011; Zhao et al., 2011; Maleci et al., 2014). The
epidermis is a direct contact barrier to protect internal tissues against
the effects of heavy metals and epidermis rupture caused by Cd prob-
ably impairs the capacity of absorption and use of macro and micro-
nutrients, generating a nutritional imbalance, consequence of the dis-
organization and reduction of cells number (De Freitas et al., 2015).
Ultrastructurally the cells of the palisade and spongy parenchyma of
S. chilensis submitted to Cd and without manure fertilization showed
drastic changes in chloroplasts, especially thylakoid disorganization
and increasing on grain starch and plastoglobuli number. These ultra-
structural changes have also been reported for chloroplasts of several
other plants exposed to heavy metals contamination (Daud et al., 2013,
2015). Chloroplasts are highly susceptible to the presence of metallic
ions in the cytoplasm (Ali et al., 2014) and this probably resulted in the

83
L.S. Pereira et al.
disruption and fragmentation of some of this organelle in S. chilensis
cells submitted to Cd. Production of starch grains in general is related to
stress on plants, such as metal contaminated environments, which in-
creases nutrient deficiency and the load on the vascular system due to
the high translocation of Cd to aerial parts (Khan et al., 2017; Daud
et al., 2015). Plastoglobuli particles of lipoprotein found inside the
chloroplasts are responsible for thylakoid membranes and proteins of
photosynthetic apparatus protection against free radicals and oxidative
stress (Austin et al., 2006). Thus, on S. chilensis species the observed
plastoglobuli represent a defense strategy to avoid damage to the
photosynthetic apparatus.
This is the first study showing the structural, nutritional and phy-
siological changes of S. chilensis cultivated in a Cd contaminated en-
vironment. Our results demonstrated that this medicinal plant species
was more tolerant to the stress caused by Cd when fertilized with a high
dose of cattle manure. This high dose of manure applied to the typical
dystrophic Ultisol resulted in a higher biomass production of S. chilensis
plants. In addition, the incorporation of the manure in the soil stimu-
lated mycorrhizal colonization in the roots of S. chilensis and the pro-
duction of glomalin in contaminated soil. The manure also promoted
the reduction of the available (soluble) Cd in the soil and the reduction
of the content of this element in the plants alleviating the effects of the
toxicity of this heavy metal on S. chilensis.
Acknowledgements
The authors would like to thank to Itamar Garcia Ignácio
(EMBRAPA Agrobiologia) for valuable help on AMF taxonomy. We also
thank to Coordination of Improvement of Higher Education Personnel
(CAPES) for the availability of the scholarship, National Council for
Scientific and Technological Development (CNPq) for funding research
and the State University of Santa Cruz (UESC) for the structure avail-
able for this work.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.ecoenv.2017.12.017.
References
Ahmad, I., Akhtar, M.J., Zahir, Z.A., Mitter, B., 2015. Organic amendments: effects on
cereals growth and cadmium remediation. Int. J. Environ. Sci. Technol. 12,
2919–2928.
Ali, B., Qian, P., Jin, R., Ali, S., Khan, M., Aziz, R., Tian, T., Zhou, W., 2014. Physiological
and ultra-structural changes in Brassica napus seedlings induced by cadmium stress.
Biol. Plant. 58, 131–138.
Ambrosini, V.G., Voges, J.G., Canton, L., Couto, R.R., Ferreira, P.A.A., Comin, J.J., Melo,
G.W.B., Brunetto, G., Soares, C.R.F.S., 2015. Effect of arbuscular mucorrhizal fungi
on young vines in copper-contaminated soil. Braz. J. Microbiol. 46, 1045–1052.
Araújo, R.P., Almeida, A.A., Pereira, L.S., Mangabeira, P.A.O., Souza, J.O., Pirovani, C.P.,
Ahnert, D., Baligar, V.C., 2017. Photosynthetic, antioxidative, molecular and ultra-
structural responses of young cacao plants to Cd toxicity in the soil. Ecotoxicol.
Environ. Saf. 144, 148–157.
Ashfaque, F., Inam, A., Inam, A., Iqbal, S., Sahay, S., 2017. Response of silicon on metal
accumulation, photosynthetic inhibition and oxidative stress in chromium-induced
mustard (Brassica juncea L.). S. Afr. J. Bot. 111, 153–160.
Austin, J.R., Frost, E., Vidi, P.A., Kessler, F., Taehelina, A.S., 2006. Plastoglobules are
lipoprotein subcompartments of the chloroplast that are permanently coupled to
thylakoid membranes and contain biosynthetic enzymes. Plant Cell. 18, 1693–1703.
Babula, P., Klejdus, B., Kovacik, J., Hedbavny, J., Hlavna, M., 2015. Lanthanum rather
than cadmium induces oxidative stress andmetabolite changes in Hypericum per-
foratum. J. Hazard. Mater. 286, 334–342.
Bedini, S., Pellegrino, E., Avio, L., Pellegrini, S., Bazzoffi, P., Argese, E., Giovannetti, M.,
2009. Changes in soil aggregation and glomalin-related soil protein content as af-
fected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus
intraradices. Soil Biol. Biochem. 41, 1491–1496.
Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,
248–254.
Brown, S.L., Chaney, R.L., Angle, J.S., Baker, A.J.M., 1994. Phytoremediation potential of
Thlaspi caerulescens and Bladder campion for zinc-contaminated and cadmium con-
taminated soil. J. Environ. Qual. 23, 1151–1157.
84
Ecotoxicology and Environmental Safety 150 (2018) 76–85
Burke, F., Hamza, S., Naseem, S., Nawaz-ul-Huda, S., Azam, M., Khan, I., 2016. Impact of
cadmium polluted groundwater on human health: winder, balochista. Sage Open.
2016, 1–8.
Carrasco, L., Azcon, R., Kohler, J., Roldán, A., Caravaca, F., 2011. Comparative effects of
native filamentous arbuscular mycorrhizal fungi in the establishment of na auto-
chthonous, leguminous shrub growing in a metal-contaminated soil. Sci. Total
Environ. 409, 1205–1209.
Chanda, D., Sharma, G.D., Jha, D.K., 2014. Isolation na identification of some Arbuscular
Mycorrhiza (AM) fungi for phytoremediation in soil contaminated with paper mill
effluent. Int. J. Curr. Microbiol. Appl. Sci. 3, 527–539.
Chen, X., Tang, J.J., Zhi, G.Y., Hu, S.J., 2005. Arbuscular mycorrhizal colonization and
phosphorus acquisition of plants: effects of coexisting plant species. Appl. Soil Ecol.
28, 259–269.
Cicatelli, A., Todeschini, V., Lingua, G., Biondi, S., Torrigiani, P., Castiglione, S., 2014.
Epigenetic control of heavy metal stress response in mycorrhizal versus non-mycor-
rhizal poplar plants. Environ. Sci. Pollut. R. 21, 1723–1737.
Daud, M.K., Shafaqat, A., Variath, M.T., Zhu, S.J., 2013. Differential physiological, ul-
tramorphological and metabolic responses of cotton cultivars under cadmium stress.
Chemosphere 93, 2593–2602.
Daud, M.K., Quiling, H., Lei, M., Ali, B., Zhu, S.J., 2015. Ultrastructural, metabolic and
proteomic changes in leaves of upland cotton in response to cadmium stress.
Chemosphere 120, 309–320.
De Andrade, S.A., da Silveira, A.P., Jorge, R.A., de Abreu, M.,F., 2008. Cadmium accu-
mulation in sunflower plants influenced by Arbuscular mycorrhiza. Int. J.
Phytoremediat. 10, 1–13.
De Freitas, T.A., França, M.G.C., de Almeida, A.-A.F., de Oliveira, S.J.R., de Jesus, R.M.,
Souza, V.L., Silva, J.V.S., Mangabeira, P.A., 2015. Morphology, ultrastructure and
mineral uptake is affected by copper toxicity in young plants of Inga subnuda subs.
luschnathiana (Benth.) T.D. Penn. Environ. Sci. Pollut. R. 22, 15479–15494.
Doubkorá, P., Sudová, R., 2016. Limited impact of arbuscular mycorrhizal fungi on clones
of Agrostis cappilaris with different heavy metal tolerance. Appl. Soil Ecol. 99, 78–88.
Dresler, S., Hanaka, A., Bednarek, W., Maksymiec, W., 2014. Accumulation of low-mo-
lecular-weight organic acids in roots and leaf segments of Zea mays plants treated
with cadmium and copper. Acta Physiol. Plant. 36, 1565–1575.
Embrapa, 2011. – Empresa Brasileira de Pesquisa Agropecuária Centro Nacional de
Pesquisas de Solos. Manual de métodos de análises de solos 2.ed. Rio de Janeiro,
Embrapa Solos, 230.
Garg, N., Bhandari, P., 2014. Cadmium toxicity in crop plants and its alleviation by ar-
buscular mycorrhizal (AM) fungi: an overview. Plant Biosyst. 148, 609–621.
Gerdemann, J.W., Nicolson, T.H., 1963. Spores of mycorrhizal Endogone species extracted
from soil by wet sieving and decanting. Trans. Br. Mycol. Soc. 46, 235–244.
Gill, S.S., Khan, N.A., Tuteja, N., 2012. Cadmium at high dose perturbs growth, photo-
synthesis and nitrogen metabolism while at low dose it up regulates sulfur assim-
ilation and antioxidant machinery in garden cress (Lepidium sativum L.). Plant Sci.
182, 112–120.
Glassman, S.I., Casper, B.B., 2012. Biotic contexts alter metal sequestration and AMF
effects on plant growth in soils polluted with heavy metals. Ecology 93, 1550–1559.
Gomes, M.P., Marques, T.C.L.L.S.M., Nogueira, M.O.G., Castro, E.M., Soares, A.M., 2011.
Ecophysiological and anatomical changes due to uptake and accumulation of heavy
metal in Brachiaria decumbens. Sci. Agric. 68, 566–573.
González-Chávez, C., Carrillo-González, R., Wright, S.F., Nichols, K.A., 2004. The role of
glomalin, a protein produced by arbuscular mycorrhizal fungi in sequestering po-
tentially toxic elements. Environ. Poll. 130, 317–323.
Hassan, S.E., Hijri, M., St-Arnaud, M., 2013. Effect of arbuscular mycorrhizal fungi on
trace metal uptake by sunflower plants grown on cadmium contaminated soil. New
Biotechnol. 30, 6.
He, S., He, Z., Yang, X., Stoffella, P.J., Baligar, V.C., 2015. Soil biogeochemistry, plant
physiology and phytoremediation of cadmium contaminated soils. In: In: Sparks, D.L.
(Ed.), Advances in Agronomy 134. pp. 135–225.
Iori, V., Pietrini, F., Massacci, A., Zacchini, M., 2014. Morphophysiological Responses,
Heavy Metal Accumulation and Phytoremoval Ability in Four Willow Clones Exposed
to Cadmium under Hydroponics. in Phytoremediation. Springer International
Publishing, Switzerland, pp. 87–98.
Jannoura, R., Joergensen, R.G., Bruns, C., 2014. Organic fertilizer effects on growth, crop
yield, and soil microbial biomass indices in sole and intercropped peas and oats under
organic farming conditions. Eur. J. Agron. 52, 259–270.
Jenkins, W.R.A., 1964. Rapid centrifugal-flotation technique for separating nematodes
from soil. Plant Dis. Rep. 48, 692–694.
Khan, A.R., Ullah, I., Waqas, M., Park, G.S., Khan, A.L., Hong, S.J., Ullah, R., Jung, B.K.,
Park, C.E., Ur-Rehman, S., Lee, I.J., 2017. Host plant growth promotion and cadmium
detoxification in Solanum nigrum, mediated by endophytic fungi. Ecotoxicol. Environ.
Saf. 136, 180–188.
Kiran, Y.K., Barkat, A., Xiao-qiang, C., Ying, F., Feng-shan, P., Lin, T., Xiao, Y., 2017. Cow
manure and cow manure-derived biochar application as a soil amendment for re-
ducing cadmium availability and accumulation by Brassica chinensis L. in acidic red
soil. J. Integr. Agric. 16, 725–734.
Kovácik, J., Babula, P., 2017. Fluorescence microscopy as a tool for visualization of metal-
induced oxidative stress in plants. Acta Physiol. Plant. 39–157.
Leal, P.L., Varón-López, M., Prado, I.G.O., Santos, J.V., Soares, C.R.F.S., Siqueira, J.O.,
Moreira, F.M.S., 2016. Enrichment of arbuscular mycorrhzal fungi in a contaminated
soil after rehabilitation. Braz. J. Microbiol. 47, 853–862.
Li, X., Zhou, Q., Sun, X., Ren, W., 2016. Effects of cadmium on uptake and translocation of
nutrient elements in different welsh onion (Allium fistulosum L.) cultivars. Food Chem.
194, 101–110.
Maleci, L., Buffa, G., Wahsha, M., Bini, C., 2014. Morphological changes induced by
heavy metals in dandelion (Taraxacum officinale Web.) growing on mine soils. Soils
L.S. Pereira et al.
Sediment. 14, 731–743.
Mcgonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.Á., 1990. A new
method which gives an objective measure of colonization of roots by vesicular-ar-
buscular mycorrhizal fungi. New Phytol. 115, 495–501.
Moreno-Jiménez, E., Fernández, J.M., Puschenreite, M., Williams, P.N., Plaza, C., 2016.
Availability and transfer to grain of As, Cd, Cu, Ni, Pb and Zn in a barley agri-system:
impact of biochar, organic and organic and mineral fertilizers. Agric. Ecosyst.
Environ. 219, 171–178.
Morton, J.B., Bentivenga, S.P., Wheeler, W.W., 1993. Germplasm in the international
collection of arbuscular and vesicular arbuscular mycorrhizal fungi (INVAM) and
procedures for culture development, documentation and storage. Mycotaxon 48,
491–528.
Mwamba, T.M., Ali, S., Ali, B., Lwalaba, J.L., Liu, H., Farooq, M.A., Shou, J., Zhou, W.,
2016. Interactive effects of cadmium and copper on metal accumulation, oxidative
stress, and mineral composition in Brassica napus. Int. J. Environ. Sci. Technol.
Najeeb, U., Jilani, G., Ali, S., Sarwar, M., Xu, L., Zhou, W., 2011. Insights into cadmium
induced physiological and ultra-structural disorders in Juncus effusus L. and its re-
mediation through exogenous citric acid. J. Hazard. Mater. 186, 565–574.
O’Brien, T.P., Feder, N., Mucully, M.E., 1964. Polycromatic stairing of plant cell walls by
toluidine blue. Protoplasma 59, 368.
Oldare, M., Arthurson, V., Pell, M., Svensson, K., Nehrenheim, E., Abubakar, J., 2011.
Land application of organic waste—effects on the soil ecosystem. Appl. Energy 88,
2210–2218.
Orowska, E., Przyby, W., Orlowski, D., Turnau, K., Mesjasz-Przyby owicz, J., 2011. The
effect of mycorrhiza on the growth and elemental composition of
Nihyperaccumulating plant Berkheya coddii Roessler. Environ. Pollut. 159,
3730–3738.
Pardo, T., Clemente, R., Bernal, M.P., 2011. Effects of compost, pig slurry and lime on
trace element solubility and toxicity in two soils differently affected by mining ac-
tivities. Chemosphere 84, 642–650.
Park, J.H., Panneerselvam, P., Lamb, D.T., Choppala, G., Bolan, N.S., Chung, J.W., 2011.
Role of organic amendments on enhanced bioremediation of heavy metal (loid)
contaminated soils. J. Hazard. Mater. 185, 549–574.
Pelfrêne, A., Waterlot, C., Mazzuca, M., Nisse, C., Cuny, D., Richard, A., Denys, S.,
Heyman, C., Roussel, H., Bidar, G., Douay, F., 2012. Bioaccessibility of trace elements
as affected by soil parameters in smelter-contaminated agricultural soils: a statistical
modeling approach. Environ. Pollut. 160, 130–138.
Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining
parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection.
Trans. Br. Mycol. Soc. 55, 158–161.
Pirovani, C.P., Carvalho, H.A.S., Machado, R.C.R., Gomes, D.S., Alvim, F.C., Pomella,
A.W.V., Gramacho, K.P., Cascardo, J.C.D.M., Pereira, G.A.G., Micheli, F., 2008.
Protein extraction for proteome analysis from cacao leaves and meristems, organs
infected by Moniliophthora perniciosa, the causal agent of the witches' broom dis-
ease. Electrophoresis 29, 2391–2401.
Ramon Junior, W.A., Piato, A.L., Conterato, G.M.M., Wildner, S.M., Marcon, M., Mocelin,
R., Emanuelli, M.P., Emanuelli, T., Nepel, A., Barison, A., Santos, C.A.M., 2015.
Hypolipidemic effects of Solidago chilensis hydroalcoholic extract and its major
85
Ecotoxicology and Environmental Safety 150 (2018) 76–85
isolated constituent quercetrin in cholesterol-fed rats. Pharm. Biol. 53, 1488–1495.
Rivera-Becerril, F., Calantzis, C., Turnau, K., Caussanel, J.P., Belimov, A.A., Gianinazzi,
S., 2002. Cadmium accumulation and buffering of cadmium-induced stress by ar-
buscular mycorrhiza in three Pisum sativum L. genotypes. Environ. Exp. Bot. 53,
1177–1185.
Rizwan, M., Ali, S., Adrees, M., Ibrahim, M., Tsang, D.C.W., Zia-ur-Rehman, M., Zahir,
Z.A., Rinklebe, J., Tack, F.M.G., Ok, Y.S., 2017. A critical review on effects, tolerance
mechanisms and management of cadmium in vegetables. Chemosphere 182, 90–105.
Russo, A., Garbarino, J., 2008. Solidago chilensis Meyen e Kageneckia oblonga Ruiz & Pav.:
petite revue de leur proful antioxydant. Phytotherapie 6, 333–341.
Saad, E.H., Mohamed, H., Marc St, A., 2013. Effect of arbuscular mycorrhizal fungi on
trace metal uptake by sunflower plants grown on cadmium contaminated soil. New
Biotechnol. 30, 780–787.
Schenck, N.C., Pérez, Y., 1988. Manual for the Identification of VA Mycorrhizal Fungi,
2nd ed. Gainesville, IFAS, University of Florida, pp. 214.
Schneider, J., Sturmer, S.L., Guilherme, L.R.G., Morira, F.M.S., Soares, C.R.F.S., 2013.
Arbuscular mycorrhizal fungi in arsenic-contaminated areas in Brazil. J. Hazard.
Mater. 262, 1105–1115.
Sun, Y., Zhou, Q., Wang, L., Liu, W., 2008. Cadmium tolerance and accumulation char-
acteristics of Bidens pilosa L. as a potential Cd-hyperaccumulator. J. Hazard. Mater.
10, 808–814.
Templeton, D.M., Liu, Y., 2010. Multiple roles of cadmium in cell death and survival.
Chem. Biol. Interact. 188, 267–275.
Verbruggen, N., Hermans, C., Schat, H., 2009. Mechanisms to cope with arsenic or cad-
mium excess in plants. Curr. Opin. Plant Biol. 12, 364–372.
Weryszko-Chmielewska, E., Chwil, M., 2005. Lead-induced histological and ultra-
structural changes in the leaves of soybean (Glycine max (L.) Merr.). Soil Sci. Plant
Nutr. 51, 203–212.
Wright, S.F., Upadhyaya, A.A., 1996. Extraction of an abundant and unusual protein from
soil and comparison with hyphal protein of arbuscular mycorrhizal fungi. Soil Sci.
161, 575–586.
Yang, Y., Song, Y., Scheller, H.V., Ghosh, A., Ban, Y., Chen, H., Tang, M., 2015.
Community structure of arbuscular mycorrhizal fungi associated with Robinia pseu-
doacacia in uncontaminated and heavy metal contaminated soil. Soil Biol. Biochem.
86, 146–158.
Zaefarian, F., Rezvani, M., Rejali, F., Ardakani, M.R., Noormohammadi, G., 2010. Ability
of Glomus mosseae- Alfalfa (Medicago sativa L.) Association for heavy metal phy-
toextraction from. Soil. Environ. Sci. 7, 77–90.
Zhang, L., Nichols, R.G., Correll, J., Murray, I.A., Tanaka, N., Smith, P.B., Hubbard, T.D.,
Sebastian, A., Albert, I., Hatzakis, E., Gonzalez, F.J., Perdew, G.H., Patterson, A.D.,
2015. Persistent organic pollutants modify gut microbiota-host metabolic home-
ostasis in mice through aryl hydrocarbon receptor activation. Environ. Health
Perspect. 123, 679–688.
Zhao, S., Ye, X., Zheng, J., 2011. Lead-induced changes in plant morphology, cell ultra-
structure, growth and yields of tomato. Afr. J. Biotechnol. 10, 10116–10124.
Zhou, S., Chen, S., Yuan, Y., Lu, Q., 2015. Influence of humic acid complexation with
metal ions on extracellular electron transfer activity. Sci. Rep. 5, 1–9.