Journal of Soils and Sediments (2020) 20:260–271

https://doi.org/10.1007/s11368-019-02414-3

SOILS, SEC 2 • GLOBAL CHANGE, ENVIRON RISK ASSESS, SUSTAINABLE LAND USE • RESEARCH
ARTICLE

A consortium of fungal isolates and biochar improved

the phytoremediation potential of Jacaranda mimosifolia D. Don
and reduced copper, manganese, and zinc leaching
Christyan Paiva Farias 1 & Gabriel Sousa Alves 1 & Denis Coelho Oliveira 2 & Edmar Isaías de Melo 3 &
Lucas Carvalho Basilio Azevedo 1

Received: 15 January 2019 / Accepted: 22 July 2019 / Published online: 7 August 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Purpose A consortium of fungal isolates may improve phytoremediation by stimulating root growth whereas biochar reduces
metal leaching in contaminated soils. We combined these treatments to evaluate to what extent Jacaranda mimosifolia D. Don
seedlings extract metals (Cu, Mn, and Zn) and reduce leaching in soil columns.
Materials and methods Contamination treatments were established by combining Cu (200 mg dm−3), Mn (450 mg dm−3), and
Zn (450 mg dm−3). A spore suspension of five fungal isolates (Beauveria bassiana, Metarhizium anisopliae, Pochonia
chlamydosporia, Purpureocillium lilacinum, and Trichoderma asperella) was sprayed on plant shoots and the soil surface.
Biochar (1% m/v) was produced by the pyrolysis of wood sawdust and applied to the soil surface. Finally, control treatments
were established in a completely randomized block design.
Results and discussion The consortium of fungal isolates increased shoot and root mass, improved the translocation potential of
Cu (translocation factor (TF) of 0.25), Mn (TF of 2.93), and Zn (TF of 1.79) from roots to shoots, and increased the total
accumulated mass of Mn and Zn in shoots and roots. Biochar applications improved Cu, Mn, and Zn translocation and increased
the total accumulated mass of shoot Mn. Moreover, a combination of fungal isolates and biochar increased the translocation
factor to 0.44 for Cu, 1.70 for Mn, and 1.34 for Zn relative to the treatment with J. mimosifolia in contaminated soil (CJ);
increased shoot Cu concentration; and reduced total leached Cu by a factor of 22.9, Mn by 5.5, and Zn by 22.9 relative to the CJ
treatment. A photochemical profile, based on chlorophyll a fluorescence, showed that Jacaranda mimosifolia was tolerant to
contaminated soil and is a potential phytoremediator of Cu, Mn, and Zn.
Conclusions Overall, the consortium of fungal isolates and biochar improved the efficiency of phytoremediation by concentrating
metals in plant tissue and reducing the risks of leaching.

Keywords Heavy metal . Phytoextraction . Plant stress . Soil contamination . Trace elements . Translocation factor

1 Introduction

The use of metals has increased, intensified, and diversified

Responsible editor: Hailong Wang since the industrial revolution because of continuous techno-
logical development (Wiedmann and Schandl 2015; Khalid
* Lucas Carvalho Basilio Azevedo
et al. 2017). Concomitantly, trace elements have been accumu-
[email protected]
lating in the environment because of mining, electroplating,
1
Institute of Agricultural Sciences, Federal University of Uberlândia, general processing, recurring pesticide and fertilizer applica-
Uberlândia, MG 38400-902, Brazil tions, car traffic, sewage, and animal waste (Wuana and
2
Institute of Biology, Federal University of Uberlândia, OKieimen 2011). Trace element contamination, even at low
Uberlândia, MG 38400-902, Brazil concentrations, affects soil productivity, ecosystem biota, fluxes
3
Institute of Chemistry, Federal University of Uberlândia, of elements and water, and human and animal health (Su 2014;
Uberlândia, MG 38400-902, Brazil Khalid et al. 2017; Sun et al. 2019).
J Soils Sediments (2020) 20:260–271 261

Unlike degradable organic substances, metals tend to accu- Jacaranda mimosifolia D. Don, we applied biochar and a con-
mulate in the environment and especially in soils (Su 2014; sortium of fungal isolates (Beauveria bassiana, Metarhizium
Zhao et al. 2019). Removing trace elements from the soil is anisopliae, Pochonia chlamydosporia, Purpureocillium
difficult due to adsorption by the soil matrix that is mainly lilacinum, and Trichoderma asperella) and assessed
influenced by soil physical-chemical properties such as the phytoextraction, leaching, and phytotoxicity of Cu, Mn, and
nature and quantity of organic matter, texture, mineralogy, Zn contamination in the soil. We hypothesized that the appli-
and pH (Dube et al. 2001; Zhao et al. 2019). Trace element cation of the consortium of fungal isolates and the biochar,
contamination in the soil can be cleaned up using physical- alone or in combination, would improve plant tolerance to trace
chemical processes; however, these are expensive and gener- elements, stimulate root growth, enhance plant uptake of Cu,
ally produce adverse environmental effects (Khalid et al. Mn, and Zn, and reduce leaching of these trace elements.
2017). Biological processes, such as plant growth, may pro-
vide alternative soil remediation strategies that are economical
and environmentally friendly (Thakur et al. 2016; Yang 2018; 2 Material and methods
Ashraf et al. 2019). Phytoremediation can accumulate and
stabilize metals in roots or shoots, while phytoextraction can 2.1 Greenhouse assay
be accomplished by harvesting the aerial part of the plant.
Physical, chemical, and biological conditioning of the soil Jacaranda mimosifolia D. Don seedlings were cultivated in
can improve plant growth and phytoremediation. For exam- polyethylene terephthalate (PET) soil columns (ø = 10 cm)
ple, plant growth promoting fungi can expand root systems filled with 1.2 dm3 of clayey Rhodic Ferralsol soil (collected
and trace element phytoextraction (Cao et al. 2008; Jaber and at 18° 57′ 34″ S; 48° 12′ 05″ W, August 2017). Prior to soil
Enkerli 2016; Feng et al. 2017; Shi et al. 2017; Farias et al. collection, the area had been used for corn (summer crop,
2018; Li et al. 2019). This stronger root growth probably 2015/2016) and sorghum (winter crop, 2016). The soil had
results from the auxins produced by the fungi (Liao et al. the following chemical properties: pH = 5.8; organic carbon =
2017; Zhang et al. 2019) or the fungi induced regulation of 22 g kg−1; Ca = 3.0 cmolc kg−1, Mg = 0.9 cmolc kg−1, K =
the plant gene involved in phytohormone synthesis (Martínez- 0.2 cmolc kg−1, Al = 0.1 cmolc kg−1, H+Al = 4.2 cmolc kg−1,
Medina et al. 2014). In addition to improving plant growth, and cation exchange capacity (CEC) = 8.3 cmolc kg−1. Based
some fungal isolates promote metal adsorption by cell wall on soil contamination values in Brazil (Conama 2009), we
functional groups or extracellular polymeric substances applied Cu (200 mg dm−3), Mn (450 mg dm−3), and Zn
(Oggerin et al. 2013; Mishra et al. 2017). (450 mg dm−3), and a consortium of fungal isolates and bio-
Biochar is another possible treatment for trace element con- char (Table 1).
tamination in the soil. Biochar improves metal remediation The J. mimosifolia seedlings were supplied by the munic-
because of its high specific area, porosity, water-holding ca- ipal garden of Uberlândia, which obtained the seeds from trees
pacity, and cation and anion adsorption, and consequently, by in urban and rural areas of the municipality. The seedlings
preventing the leaching of nutrients and metals (Cao and were grouped in six blocks according to height (average
Harris 2010; Uchimiya et al. 2010; Zhang et al. 2013; Wang 33.6 cm from root–shoot intersection to the first leaf inser-
et al. 2019a, b). In addition to reducing metal leaching, biochar tion). The seedlings were washed to remove substrate from
can also decrease plant stress by reducing metal bioavailabil- the roots and then transplanted into soil columns. The follow-
ity and the phytotoxicity of contaminated soils (Bandara et al. ing fertilizers were applied to the soil surface: calcium carbon-
2017; Gong et al. 2019a). Therefore, phytoremediation strat- ate (500 mg dm−3), nitrogen (300 mg dm−3), phosphorus
egies may benefit from enhancing plant tolerance to metal- (300 mg dm −3 ), potassium (600 mg dm −3), and sulfur
contaminated soil. Plant tolerance to metal contamination (80 mg dm−3). Each plot was irrigated daily with 100 mL of
can be monitored using photosynthesis parameters such as tap water (pH = 7.26; heterotrophic bacteria = 2.0 CFU mL−1;
chlorophyll pigment concentration and chlorophyll a fluores- coliforms and Escherichia coli absent; residual Cl =
cence of photosystem II (Vaillant et al. 2005; Janik et al. 2010; 0.3 mg L −1 ; surfactants < 0.05 mg L −1 ; turbidity =
Cambrollé 2015; Gong et al. 2019b). 1.39 NTU; Sb, Cd, and Hg < 0.001 mg L−1; As and Se <
Thus, fungal isolates and biochar, either alone or combined, 0.005 mg L−1; Pb < 0.006 mg L−1; Cu < 0.008 mg L−1; U <
can improve plant growth by increasing the uptake and trans- 0.01 mg L−1). The assay was conducted for 45 days until
location of trace element from roots to the shoots, which in turn harvesting.
facilitates phytoremediation (Khan et al. 2017; Shi et al. 2017;
Gong et al. 2019a). However, there is a lack of studies on 2.2 Inoculation of the fungal isolate consortium
beneficial plant–fungal interactions with biochar to improve
the phytoremediation and avoid the leaching of trace elements. A consortium of five fungal isolates was applied to the soil
Therefore, to evaluate the phytoremediation potential of surface and sprayed twice on the shoots in some treatments
262 J Soils Sediments (2020) 20:260–271

Table 1 Treatments for

phytoremediation assay in soil Treatment Code
column contaminated with Cu,
Mn, and Zn, application of the Non-contaminated soil NC
consortium of five fungal isolates Non-contaminated soil with Jacaranda mimosifolia NCJ
or the biochar Contaminated soil1 C
Contaminated soil1 with Jacaranda mimosifolia CJ
Contaminated soil1 with Jacaranda mimosifolia and fungal consortium2 CJF
Contaminated soil1 with Jacaranda mimosifolia and biochar3 CJB
Contaminated soil1 with Jacaranda mimosifolia fungal consortium2 and biochar3 CJFB

Assay established in a completely randomized block design with six replicates

1
Contaminated soil with Cu (200 mg dm−3 ), Mn (450 mg dm−3 ), and Zn (450 mg dm−3 )
2
Five fungal isolates application: Beauveria bassiana, Metarhizium anisopliae, Pochonia chlamydosporia,
Purpureocillium lilacinum, and Trichoderma asperella
3
Biochar obtained from slow pyrolysis of sawdust wood (Citriodora sp.)

(Table 1). Before the applications, the fungal isolates 2.4 Stress measurement using photosynthetic
(Beauveria bassiana, Metarhizium anisopliae, Pochonia parameters
chlamydosporia, Purpureocillium lilacinum, and
Trichoderma asperella) were stored in flasks (BIOSAG: Quenching analysis (assessed by chlorophyll a fluorescence)
Comércio e Serviços Agrícolas Ltda). The individual isolates was performed 43 days after transplanting using a modulated
in each flask were homogenized in 500 mL of distilled water fluorescence imaging apparatus (Handy FluorCam PSI, Photo
using a blender and spore counts were determined using a Systems Instrument, Czech Republic). Electron chain integri-
Neubauer chamber. Next, the five fungal isolate suspensions ty and the capacity of the plant tissue to photosynthesize and
were combined into a total volume of 2.5 L with the following to withstand metal contamination were demonstrated after a
spore densities: 4.20 × 106 mL−1 Beauveria bassiana; 7.45 × dark-adapted period (20 min) and exposure to various light
106 mL−1 Metarhizium anisopliae; 5.35 × 106 mL−1 Pochonia treatments according to the Quenching software protocol
chlamydosporia; 3.65 × 106 mL−1 Purpureocillium lilacinum; (Photo Systems Instruments, Version 2) (Oliveira et al.
and 3.12 × 106 mL−1 Trichoderma asperella. 2017). The following parameters were used: F0 (minimum
Twenty milliliters of this suspension was applied to the soil fluorescence of PSII in a dark-adapted state); Fm (maximum
surface of each column. The suspension was then diluted fluorescence of PSII in a dark-adapted state); Fv/Fm (maxi-
(1:100) and 5 mL was applied to the leaves of each seedling mum PSII quantum yield in a dark-adapted state, where
after transplanting. A second foliar application of 5 mL was Fv = Fm – F0); (F′m–F′)/F′m (PSII operating efficiency, where
performed 28 days later. F′m is the fluorescence signal when all PSII centers are closed
in the light-adapted state and F′ is the measurement of the
light-adapted fluorescence signal); Rfd (instantaneous fluores-
2.3 Biochar application cence decline ratio in light) and NPQ (instantaneous non-
photochemical quenching during dark relaxation).
The biochar was produced by slow pyrolysis of eucalyptus Chlorophyll pigment concentration was assessed using a
(Citriodora sp.) sawdust using a homemade two-cylinder SPAD – 502 chlorophyll meter (Minolta) and expressed in
reactor (Prakongkep et al. 2015) at 350–400 °C and with SPAD values.
limited O2. The zero point charge (ZPC) of the biochar was
7.17. ZPC was measured twice by adding 20 mg of biochar 2.5 Plant growth
to 20 mL of 0.1 mol L−1 aqueous KCl solution at different
initial pH values (1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 8.00, The fresh and dry masses of J. mimosifolia shoots and roots
9.00 10.00, 11.00, 12.00), adjusted with HCl or NaOH were evaluated 45 days after transplanting to the soil column.
0.1 mol L−1 solutions. The final pH of the solutions was Dry weight was measured (analytical balance) after drying the
determined after stirring for 24 h at room temperature and plant tissue at 60 °C to a constant mass.
filtering (OH et al. 2012). A ZPC of 7.17 indicates that the
biochar produces a net positive charge (anionic exchange 2.6 Cu, Mn, and Zn contents in the plants
capacity) when added to an environment with a pH lower
than 7.17. The biochar treatments consisted of 1% (m/v) Cu, Mn, and Zn concentrations were determined in shoots and
applications to the soil surface. roots via nitropercloric digestion and subsequent
J Soils Sediments (2020) 20:260–271 263

measurement with an atomic absorption spectrophotometer reduction because of correlation with Fm and because of its
(Agilent 55B AA Spectrometer, detection limit of limited contribution to variation. Finally, (Fm′-F′)/Fm′, Fm, F0,
0.014 mg L − 1 for Cu, 0.057 mg L − 1 for Mn, and and Rfd were used in a multivariable linear mixed model
0.001 mg L−1 for Zn) (Silva 2009). (MLM). The treatments were considered corrected while in-
dividual numbers and blocks were considered as aleatory ef-
2.7 Bioconcentration factor and translocation factor fects. SPAD differences were determined using a simple
MLM in which the blocks were treated as aleatory effects.
The efficiency of metal removal from soil was measured as the We evaluated the homogeneity of fitted versus residual values,
bioconcentration factor (BF = plant metal concentration/soil quantile–quantile plots, histograms, and Cook’s distance for
metal concentration) and the translocation factor (TF = shoot validating linear models (Zuur et al. 2009). PCA was per-
metal concentration/root metal concentration). formed using FactoMineR (Husson et al. 2017) while
LMMs were created using lme4 (Bates et al. 2018) and car
2.8 Total Cu, Mn, and Zn accumulated mass (Fox et al. 2015). All PCA analyses were conducted in R
in the plants version 3.4.3 (R Development Core Team 2017).

The total mass of accumulated metals was calculated by mul-

tiplying the metal concentration by the dry mass of the plant 3 Results
tissue (shoot or root).
3.1 Stress measurement using photosynthesis
2.9 Total Cu, Mn, and Zn leached mass parameters

The leachate from the soil columns was collected daily and F0 (minimum fluorescence of PSII in a dark-adapted state)
preserved by adding HNO3 until reaching pH < 3.0. The solu- was the only photosynthetic parameter that was differenti-
tion was then filtered (blue stripe filter, Ø = 11 μm) and mea- ated among the treatments: plants in contaminated soil (CJ)
sured by inductively coupled plasma-optical emission spec- and in contaminated soil with fungi and biochar (CJFB)
trometry (ICP-OES 5100, Agilent Technologies; quantifica- produced lower values (Table 2). PCA showed that (Fm′-
tion limit of 0.0005 mg L −1 for Cu and Mn, and of F′)/Fm′, Fm, F0, and Rfd were the best explanatory variables
0.002 mg L−1 for Zn). The total mass of accumulated metals in the first and second principal components (71% of total
was calculated by multiplying the concentration of leached variability) (Fig. 1). Although (Fm′-F′)/Fm′, Fm, F0, and Rfd
metal by the total volume produced during the 45-day assay. explained most of the data variation, PCA did not discrim-
inate among the treatments (Fig. 2). MLM showed no sig-
2.10 Statistics nificant differences among the treatments (χ2 = 7.53; df =
4; p = 0.111), while the univariate MLMs of the six photo-
The assay was set up in a completely randomized block design synthesis variables were similar (data not shown). SPAD
with six replicates. ACTION 2.9 software was used to deter- also did not differentiate among the treatments (χ2 = 3.86;
mine outliers and perform the Levene homogeneity of vari- df = 4; p = 0.426).
ance test (p > 0.05) and the Anderson-Darling normality test
(p > 0.05). The data were normalized as follows: root Zn con- 3.2 Plant growth
centration, total Mn accumulated in shoots, TF of Cu and TF
of Mn were transformed by x0.5; FT was transformed by 1/x0.5; The fresh and dry root masses of J. mimosifolia grown in
and total Cu, Mn, and Zn leached masses were transformed by contaminated soils and treated with a consortium of fungal
log10. The variables were then submitted to analysis of vari- isolates (CJF and CJFB) were statistically similar to those of
ance (Levene test, p < 0.05) and the Scott-Knott test (p < 0.05) the plants in the non-contaminated control (NCJ), and higher
(Sisvar software) (Ferreira 2011). than those grown in contaminated soil (CJ) and in contami-
Pearson correlations (Proc Corr procedure, SAS Institute, nated soil with biochar (CJB) (Table 3).
Inc., Cary, North Carolina) were used to investigate cause–
effect relationships among variables showing greater 3.3 Plant Cu, Mn, and Zn concentrations
phytoremediation efficiency, with other explanatory variables.
Principal component analysis (PCA) was performed to Applications of fungi and biochar on contaminated soil
evaluate associations among photosynthesis variables and to (CJFB) increased shoot Cu concentration (Table 3).
reduce overparametrization. Fv/Fm was removed due to col- However, shoot Mn and Zn concentration did not differ
linearity with Fm (correlation = 0.69) and (Fm′-F′)/Fm′ (corre- among any of the treatments in contaminated soil. Fungal
lation = 0.74). NPQ was also removed after dimensionality and biochar applications on contaminated soil (CFJ, CJB,
264 J Soils Sediments (2020) 20:260–271

Table 2 Fluorescence and content chlorophyll variables of Jacaranda mimosifolia D. Don grown on soil columns in a greenhouse assay

Treatments F0 Fm Fv/Fm NPQ Rfd (Fm′-F′)/Fm′ Chlorophyll concentration

NCJ 58.13 (3.93) a 233.21 (32.79) a 0.75 (0.02) a 0.38 (0.14) a 3.18 (0.56) a 0.47 (0.02) a 26.39 (7.59) a
CJ 51.52 (1.44) b 253.13 (27.98) a 0.78 (0.01) a 0.34 (0.10) a 3.12 (0.75) a 0.53 (0.05) a 30.38 (8.83) a
CJF 55.47 (6.54) a 245.85 (33.92) a 0.77 (0.02) a 0.29 (0.05) a 3.36 (0.15) a 0.50 (0.04) a 30.64 (4.34) a
CJB 61.51 (5.09) a 250.21 (35.09) a 0.75 (0.04) a 0.32 (0.12) a 3.28 (0.32) a 0.48 (0.07) a 30.30 (9.25) a
CJFB 48.49 (3.87) b 205.41 (32.88) a 0.74 (0.05) a 0.25 (0.09) a 2.66 (0.34) a 0.50 (0.09) a 18.65 (3.50) a

Average (standard deviation) (n = 6). Different lowercase letters in columns indicate statistic difference (p < 0.05, Scott-Knott test). Chlorophyll con-
centration measured using a chlorophyll meter SPAD- 502. Fungal consortium consisted of five fungal isolates application: Beauveria bassiana,
Metarhizium anisopliae, Pochonia chlamydosporia, Purpureocillium lilacinum, and Trichoderma asperella. Biochar obtained from slow pyrolysis of
sawdust wood (Citriodora sp.)
NCJ, non-contaminated soil with J. mimosifolia; CJ, contaminated soil (Cu, 200 mg dm−3 ; Mn, 450 mg dm−3 ; and Zn, 450 mg dm−3 ) with
J. mimosifolia; CJF, contaminated soil with J. mimosifolia and application of the consortium of five fungal isolates; CJB, contaminated soil with
J. mimosifolia and biochar; CJFB, contaminated soil with J. mimosifolia, application of the consortium of five fungal isolates and the biochar; F0,
minimum fluorescence yield in dark-adapted state; Fm, maximum fluorescence yield in dark adaptation state; Fv/Fm, maximum quantum yield; NPQ,
non-photochemical quenching; Rfd, ratio of fluorescence decrease in stationary state; (Fm′-F′)/Fm′ PSII operational efficiency

and CJFB) reduced root Cu concentrations to the same 3.4 Bioconcentration and translocation factors
level of the control (uncontaminated), which was lower
than the contaminated soil without fungal isolates and bio- The BFs of Cu (15.1) and Zn (13.9) were higher in plants
char (CJ) treatments (Table 3). The biochar treatments grown in soil without Cu, Mn, and Zn applications (NCJ) than
(CJB and CJFB) also reduced root Mn concentrations to in plants grown in soil with Cu, Mn, and Zn applications (CJ,
the level of the uncontaminated treatment. Root Zn con- CJF, CJB, CJFB) (Table 3). Manganese BF did not differ
centrations were higher in all contaminated soil treatments among treatments. However, BF values of plants grown in
than in the control (NCJ). contaminated soil were higher than 1.0 (ranging from 1.0 to

Fig. 1 PCA biplot showing the contribution of variables and five contaminated soil with Jacaranda mimosifolia, an application of the
treatment groups. Ellipses comprise 0.95 confidence intervals. NCJ, consortium of fungal isolates and biochar. The consortium of fungal
uncontaminated soil with Jacaranda mimosifolia; CJ, contaminated soil isolates consisted of five fungal isolates: Beauveria bassiana,
(Cu, 200 mg dm−3; Mn, 450 mg dm−3; and Zn, 450 mg dm−3) with Metarhizium anisopliae, Pochonia chlamydosporia, Purpureocillium
Jacaranda mimosifolia; CJF, contaminated soil with Jacaranda lilacinum, and Trichoderma asperella. Biochar was obtained from slow
mimosifolia and an application of the consortium of fungal isolates; pyrolysis of wood sawdust (Citriodora sp.)
CJB, contaminated soil with Jacaranda mimosifolia and biochar; CJFB,
J Soils Sediments (2020) 20:260–271 265

Fig. 2 Total accumulated mass of

Cu, Mn, and Zn in the shoots and
roots of Jacaranda mimosifolia
(average, n = 6). Different
lowercase letters indicate
statistical difference (p < 0.05,
Scott-Knott test). NCJ, uncon-
taminated soil with Jacaranda
mimosifolia; CJ, contaminated
soil (Cu, 200 mg dm−3; Mn,
450 mg dm−3; and Zn,
450 mg dm−3) with Jacaranda
mimosifolia; CJF, contaminated
soil with Jacaranda mimosifolia
and an application of the consor-
tium of fungal isolates; CJB,
contaminated soil with Jacaranda
mimosifolia and biochar; CJFB,
contaminated soil with Jacaranda
mimosifolia, an application of the
consortium of fungal isolates and
biochar. The consortium of fungal
isolates consisted of five fungal
isolates: Beauveria bassiana,
Metarhizium anisopliae,
Pochonia chlamydosporia,
Purpureocillium lilacinum, and
Trichoderma asperella. Biochar
was obtained from slow pyrolysis
of wood sawdust (Citriodora sp.)

2.0 for Cu, from 2.4 to 4.4 for Mn, and from 3.7 to 6.2 for Zn) (CJFB) increased TF by a factor of 4.00 for Cu, 2.83 for
and did not differ among the treatments. Mn, and 4.47 for Zn relative to CJ.
The TF ranged from 0.11 to 0.44 for Cu, from 0.43 to 2.93
for Mn, and from 0.30 to 1.79 for Zn (Table 3). Relative to 3.5 Total accumulated mass of Cu, Mn, and Zn
plants grown in untreated contaminated soil (CJ), the consor-
tium of fungal isolates (CJF) increased TF by a factor of 2.27 Total accumulated mass (concentration×mass) of metal dif-
for Cu, 4.88 for Mn, and 5.97 for Zn. The biochar application fered from the metal concentration in the plants (Fig. 2). As
(CJB) increased TF by a factor of 2.18 for Cu and 2.00 for Zn, expected, the uncontaminated control treatment (NCJ) had the
while the combined fungal isolates and biochar treatment lowest total accumulation of shoot and root Cu, Mn, and Zn.
266 J Soils Sediments (2020) 20:260–271

Table 3 Plant growth, metal content, bioconcentration factors (BF), and translocation factors (TF) of Jacaranda mimosifolia D. Don seedlings grown
in metal-contaminated soil under application of consortium with five fungal isolates and biochar

Treatment NCJ CJ CJF CJB CJFB

Plant growth
Shoot fresh mass (g) 22.03 (2.07) a 16.96 (4.55) b 24.74 (3.36) a 19.73 (6.90) b 25.28 (0.70) a
Root fresh mass (g) 16.64 (5.48) a 10.30 (0.27) b 20.71 (5.07) a 12.57 (3.03) b 17.48 (4.86) a
Shoot dry mass (g) 13.76 (2.21) a 10.37 (1.97) b 13.51 (1.81) a 11.38 (2.67) b 14.10 (0.99) a
Root drya mass (g) 13.48 (4.06) a 8.46 (1.18) b 14.05 (2.34) a 10.34 (2.42) b 12.94 (2.55) a
Metal content
Cu shoots (mg kg−1) 6.97 (1.95) b 12.46 (2.22) b 11.83 (3.82) b 12.41 (4.93) b 19.19 (10.63) a
Mn shoots (mg kg−1) 27.8 (5.2) b 101.1 (46.6) a 137.9 (32.2) a 109.2 (37.3) a 140.2 (82.4) a
Zn shoots (mg kg−1) 16.5 (1.3) b 80.2 (22.0) a 114.6 (42.5) a 92.1 (30.5) a 107.1 (74.6) a
Cu roots (mg kg−1) 44.8 (36.2) b 127.5 (46.6) a 54.07 (20.97) b 53.60 (10.58) b 43.5 (14.2) b
Mn roots (mg kg−1) 77.4 (28.5) b 200.5 (85.6) a 232.7 (225.3) a 110.0 (16.7) b 86.8 (51.8) b
Zn roots (mg kg−1) 28.4 (26.7) b 317.3 (118.0) a 205.5 (203.9) a 156.7 (48.4) a 92.5 (33.0) a
Bioconcentration factor (BF)
Cu BF 15.14 (14.45) a 2.00 (0.64) b 1.06 (0.36) b 1.09 (0.29) b 1.01 (0.38) b
Mn BF 3.68 (1.55) a 4.38 (2.48) a 3.11 (1.96) a 3.72 (2.22) a 2.41 (1.80) a
Zn BF 13.89 (8.35) a 6.16 (1.67) b 5.28 (3.39) b 4.77 (1.83) b 3.65 (1.53) b
Translocation factor (TF)
Cu TF 0.33 (0.31) a 0.11 (0.06) b 0.25 (0.11) a 0.24 (0.10) a 0.44 (0.18) a
Mn TF 0.43 (0.25) b 0.60 (0.36) b 2.93 (3.55) a 0.98 (0.26) b 1.70 (0.83) a
Zn TF 0.89 (0.53) a 0.30 (0.17) b 1.79 (2.03) a 0.60 (0.12) a 1.34 (1.24) a

Average (standard deviation) (n = 6). Different lowercase letters in rows indicate statistic difference (p < 0.05, Scott-Knott test). Highest values are shown
in italics. Fungal consortium consisted of five fungal isolates application: Beauveria bassiana, Metarhizium anisopliae, Pochonia chlamydosporia,
Purpureocillium lilacinum, and Trichoderma asperella. Biochar obtained from slow pyrolysis of sawdust wood (Citriodora sp.)
NCJ, non-contaminated soil with J. mimosifolia; CJ, contaminated soil (Cu, 200 mg dm−3 ; Mn, 450 mg dm−3 ; and Zn, 450 mg dm−3 ) with
J. mimosifolia; CJF, contaminated soil with J. mimosifolia and PGPF consortium; CJB, contaminated soil with J. mimosifolia and biochar; CJFB,
contaminated soil with J. mimosifolia, application of the fungal consortium and the biochar

Total accumulated shoot Cu was 206% greater in the consor- biochar treatments (CJB and CJFB) than in plants grown
tium of fungal isolates and biochar treatment (CJFB) than in in contaminated soil without any applications (CJ and
the contaminated treatment without a consortium of fungal CJF).
isolates and biochar (CJ) (Fig. 2a). Accumulated root Cu
was lower in the biochar/contaminated soil treatments (CJB
and CJFB) than in the contaminated soil treatment (CJ) and in 3.6 Total leached mass of Cu, Mn, and Zn
the consortium of fungal isolates treatment (CJF) (Fig. 2a).
The total accumulated mass of shoot Mn was 1.76 times Total Mn and Zn leachate values were highest in the
greater in the consortium of fungal isolates treatment (CJF) contaminated-soil columns (C, CJ, CJF, and CJB) (Table 4).
and 1.87 times greater in the consortium of fungal isolates plus Total Cu leachate values were higher in the treatment with
biochar treatment (CJFB) than in untreated plants grown in J. mimosifolia grown in contaminated soil (CJ) and the con-
contaminated soil (CJ) (Fig. 2b). The consortium of fungal sortium of fungal isolates treatment (CJF). Cu leaching was
isolates (CJF) also enhanced the total accumulated mass of greater in the treatment with J. mimosifolia grown in contam-
root Mn by a factor of 2.03. inated soil (CJ) than in the treatment with contaminated soil
Relative to plants grown in contaminated soil without but no plants (C). However, the combined application of the
applications (CJ), the total accumulated mass of shoot Zn consortium of fungal isolates and biochar (CJFB) reduced the
was 1.84 times greater in the consortium of fungal isolates total leached amount of Cu by a factor of 22.9, Mn by 5.5, and
treatment (CJF), 1.23 times greater in the biochar treat- Zn by 22.9 relative to the treatment with J. mimosifolia in
ment (CJB), and 1.76 times greater when the consortium contaminated soil (CJ). This result indicates the potential of
of fungal isolates and biochar were applied together (Fig. combined consortium of fungal isolates/biochar applications
2c). Total accumulated mass of root Zn was lower in the in reducing the quantity of metals leached to the groundwater.
J Soils Sediments (2020) 20:260–271 267

Table 4 Total leached mass of Cu, Mn, and Zn in soil column with Mn, and Zn were positively correlated with root fresh mass,
growth of Jacaranda mimosifolia D. and application of the consortium of
while the total masses of shoot Cu, Mn, and Zn were positive-
five fungal isolates and the biochar
ly correlated with fresh shoot mass. Total root Cu, Mn, and Zn
Treatment Cu Mn (μg) Zn Volume of masses were positively correlated with the concentration of
leachate (mL) organic carbon. However, the total leached masses of Cu,
NC 9.0 b 82.5 b 98.4 c 1157 a
Mn, and Zn were not correlated with any of these explanatory
NCJ 4.0 c 9.8 c 25.0 c 285 b
variables.
C 15.5 b 6791.5 a 19,324.9 a 1408 a
CJ 563.0 a 1457.6 a 3773.3 a 1375 a
CJF 678.4 a 1500.9 a 6670.0 a 1264 a
4 Discussion
CJB 268.1 a 1506.1 a 1902.0 b 615 b
Applications of fungi, biochar, and a combination of both
CJFB 24.6 b 266.4 b 164.5 c 388 b
improved the phytoremediation efficiency of J. mimosifolia
Average (standard deviation) (n = 6). Different lowercase letters in col- and reduced metal leaching (Table 3, Fig. 2). Higher root
umns indicate statistic difference (p < 0.05, Scott-Knott test). Fungal con- and shoot mass may explain how the consortium of fungal
sortium of five fungal isolates application: Beauveria bassiana, isolates contributed to greater Zn translocation and accumula-
Metarhizium anisopliae, Pochonia chlamydosporia, Purpureocillium
lilacinum, and Trichoderma asperella. Biochar obtained from slow py- tion of shoot Mn and Zn (Table 5). These variables were
rolysis of sawdust wood (Citriodora sp.) positively correlated with TF and total accumulated mass of
NC, non-contaminated soil; NCJ, non-contaminated soil with shoot Cu, Mn, and Zn. Even though Zn contamination nor-
J. mimosifolia; C, contaminated soil; CJ, contaminated soil (Cu, mally reduces root growth (Vaillant et al. 2005), fungal inoc-
200 mg dm −3 ; Mn, 450 mg dm−3 ; and Zn, 450 mg dm−3 ) with ulation resulted in higher root mass. This same consortium of
J. mimosifolia; CJF, contaminated soil with J. mimosifolia and applica-
tion of the consortium of five fungal isolates; CJB, contaminated soil with fungal isolates also affected root growth in soybeans and corn
J. mimosifolia and biochar; CJFB, contaminated soil with J. mimosifolia, (Farias et al. 2018).
application of the consortium of five fungal isolates and the biochar T h e f u n g i M e t a rh i z i u m a n i s o p l i a e , P o c h o n i a
chlamydosporia, Purpureocillium lilacinum, and
3.7 Explanation of enhanced phytoremediation Trichoderma asperella improve plant growth in various ways
(Sasan and Bidochka 2012; Gibson et al. 2014; Liao et al.
All variables that indicated enhanced phytoremediation 2014). For example, they can induce systemic resistance in
(higher shoot Cu concentration; TF and total accumulated plants (Brotman et al. 2012; Hermosa et al. 2013; Vinayarani
mass of Cu, Mn, and Zn; and lower total leached mass of et al. 2019); affect the synthesis of jasmonic, salicylic, and
Cu, Mn, and Zn) were tested for Pearson correlations with abscisic acid (ABA) (Contreras-Cornejo et al. 2011;
explanatory variables (Table 5). Shoot Cu concentration was Martínez-Medina et al. 2011); and expand root systems
positively correlated with K concentration. The TFs of Cu, (Jaber and Enkerli 2016; Farias et al. 2018). Auxins produced

Table 5 Pearson correlation among phytoremediation, plant growth, and soil chemical variables under growth of Jacaranda mimosifolia D. in soil
columns

Response variable Shoot fresh mass Root fresh mass Shoot dry mass Root dry mass pH Org. C CEC K

Shoots Cu content 0.31 0.06 0.24 − 0.02 0.03 − 0.03 − 0.08 0.40*
TF Cu 0.50** 0.49** 0.57** 0.48** − 0.05 − 0.21 − 0.00 0.12
TF Mn 0.24 0.49** 0.15 0.38* − 0.02 − 0.28 − 0.00 0.18
TF Zn 0.24 0.38* 0.18 0.33 0.01 0.18 0.01 0.48
Total shoot Cu 0.51** 0.24 0.46** 0.17 0.05 0.04 0.05 0.44*
Total shoot Mn 0.44* 0.35 0.32 0.26 0.13 0.23 0.16 0.60***
Total shoot Zn 0.45* 0.30 0.31 0.20 0.01 0.15 0.04 0.45*
Total root Cu − 0.18 − 0.14 − 0.25 − 0.14 0.10 0.44* 0.26 0.09
Total root Mn 0.13 0.19 0.04 0.23 0.11 0.49* 0.54** 0.32
Total root Zn 0.09 0.06 − 0.12 0.02 0.07 0.45* 0.40* 0.29
Total leached Cu − 0.13 − 0.08 − 0.20 − 0.08 − 0.18 0.16 0.09 0.03
Total leached Mn − 0.31 − 0.14 − 0.34 − 0.18 0.10 0.01 0.03 0.07
Total leached Zn − 0.14 − 0.03 − 0.20 − 0.04 − 0.19 0.20 0.17 0.11

Italicized values indicate significant Pearson correlations: *p < 0.05; **p < 0.01; ***p < 0.001
268 J Soils Sediments (2020) 20:260–271

by these fungi may be responsible for expanding the root in turn decreases plant uptake and translocation to the shoots
system (Contreras-Cornejo et al. 2009; Cavello et al. 2015; (Kim et al. 2015; Bandara et al. 2017; Gong et al. 2019a).
Zavala-Gonzalez et al. 2015; Liao et al. 2017; Bader et al. Nevertheless, our TF findings for Cu, Mn, and Zn indicate
2019) or regulating the plant synthesis of auxins (Contreras- that the consortium of fungal isolates and biochar applications
Cornejo et al. 2011; Khan et al. 2012; Martínez-Medina et al. on J. mimosifolia improve metal phytoextraction.
2014). Trichoderma can also induce ABA synthesis in plants In addition to phytoextraction, the combined application of
(Raghavendra et al. 2010; Ramírez-Valdespino et al. 2019) via biochar and fungi (CJFB) increased the total accumulated
a phytohormone that promotes growth under abiotic stress. mass of shoot Cu and reduced the total mass of leached Cu,
Thus, the improvements in Cu, Mn, and Zn translocation Mn, and Zn (Table 4). The same was not observed when the
and accumulation in J. mimosifolia shoots brought about by consortium of fungal isolates and biochar were applied sepa-
the consortium of fungal isolates may result from the regula- rately, indicating that these conditioners are interacting with
tion of phytohormone synthesis (auxin and ABA) that in turn one another. Higher accumulated Cu could be directly or in-
increases root growth, shoot mass, and metal uptake while directly related to K concentration in the soil (Table 5).
decreasing leachates (when combined with biochar). Studies However, some studies have shown that K concentration has
reporting on correlations between root biomass and the trans- no effect on Cu binding sites in the roots, plant uptake or
location of Cu, Mn, and Zn to shoots and reductions in Cu, phytotoxicity (Lamb et al. 2012; Chen et al. 2013).
Mn, and Zn leaching are scarce. The combined consortium of fungal isolates and biochar
Bioconcentration factors tend to be higher than 1.0 in soils treatment could be used to decrease the risks of groundwater
with lower metal concentrations, as in the soils without Cu, contamination by reducing total leachate mass. This reduction
Mn, and Zn applications (NCJ). This happened mainly be- may result from an expanded root system with a greater capac-
cause these metals are plant nutrients and occurred at low ity for Cu, Mn, and Zn uptake (Table 3), cation uptake, and
concentrations. Thus, attention must be paid to the treatments water retention provided by biochar (Cao and Harris 2010;
that received these metals as contaminants. Therefore, BF can Uchimiya et al. 2010; Zhang et al. 2013; Wang et al. 2019a,
be used to select plants suitable for phytoextraction (McGrath b). Biochar can change pH, cation exchange capacity (CEC),
and Zhao 2003; Lam et al. 2018; Ameh et al. 2019). BF was and organic matter concentration, which in turn influence metal
greater than 1.0 in the contaminated soil treatments, suggest- retention and leaching susceptibility (Zhang et al. 2013; Kargar
ing that J. mimosifolia may be an effective phytoextractor for et al. 2015; Wang et al. 2019a, b). However, our correlation
Cu, Mn, and Zn (Yoon et al. 2006; Buscaroli 2017; Lam et al. analysis did not show that a reduction in leaching was ex-
2017, 2018). BF did not vary significantly among the treat- plained by plant mass or soil chemical variables such as pH,
ments with only J. mimosifolia or in combination with the CEC, or organic carbon (Table 5). Biochar by itself (CJB) or
consortium of fungal isolates and biochar. combined with the consortium of fungal isolates (CJFB) re-
In addition to bioconcentration, metal translocation from duced the total leached volume produced during the assay
roots to shoots is another important trait of plants used for (Table 4); however, the mass of leached metal was only reduced
remediation (Yoon et al. 2006; Ameh et al. 2019). The TF is by the combined application (CJFB). Thus, the consortium of
especially important for selecting plants that will be harvested fungal isolates and biochar combined may cause metal adsorp-
to remove metals from contaminated sites (Thakur et al. 2016; tion in cell wall functional groups and in extracellular polymer-
Ameh et al. 2019). We demonstrated that a consortium of ic substances (Oggerin et al. 2013; Mishra et al. 2017).
fungal isolates improves the capacity of J. mimosifolia to In addition to reducing environmental risks, phytoremediators
transfer Cu, Mn, and Zn from roots to the shoots. must also function under stressful conditions in order to provide
Specifically, the TF of Cu in J. mimosifolia increased from effective remediation in the field. An analysis of chlorophyll a
0.11 to 0.25 with the consortium of fungal isolates, and to 0.44 fluorescence of photosystems II can reveal abiotic stress in
when combined with biochar, while the TF of Mn changed plants, including stress caused by trace elements (Vaillant et al.
from 0.60 to 2.93, and the TF of Zn rose from 0.30 to 1.79. 2005; Cambrollé 2015; Gong et al. 2019b). Lower Fv/Fm and
Other studies have reported that endophytic microorganisms higher F0 are commonly associated with plant responses to stress
can increase TF (Cao et al. 2008; Shi et al. 2017; Chao et al. (Murchie and Lawson 2013). Moreover, decreases in (F′m–F′)/F
2019) or decrease TF after endophyte fungal or bacterial in- ′m and Rfd can also indicate plant stress (Lichtenthaler and Miehé
oculations (Ma et al. 2016; Chen et al. 2017). However, little 1997). In our study, chlorophyll fluorescence did not differ
is known about plant physiology and TF modulation resulting among treatments (Fig. 2 and Table 2), indicating that metal
from inoculations with beneficial microorganisms. contamination did not affect the photosystem II reaction center
While biochar applications improve the TF of Cu, Mn, and (PS II) of J. mimosifolia (Bolhar-Nordenkampf et al. 1989). One
Zn, they have also been shown to reduce the TF of metals (Ali exception was F0, which was lower in contaminated soil (CJ)
et al. 2017; Khan et al. 2017; Eissa 2019). This happens be- than that in non-contaminated soil (NCJ). Overall, our results
cause biochar reduces metal bioavailability in the soil, which indicate that J. mimosifolia has potential for phytoremediation.
J Soils Sediments (2020) 20:260–271 269

5 Conclusions Ameh EG, Omatola OD, Akinde SB (2019) Phytoremediation of toxic

metal polluted soil: screening for new indigenous accumulator and
translocator plant species, northern Anambra Basin, Nigeria.
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improving plant growth, translocation factors, and total accu- Phytoremediation: environmentally sustainable way for reclamation
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mulated mass of shoot Cu, Mn, and Zn and reducing total mass
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the translocation potential of Cu, Mn, and Zn from roots to the on tomato (Solanum lycopersicum L.). J King Saud Univ Sci.
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Acknowledgments Authors thank the reviewers for their comments and tinent to its potential use in remediation. Bioresour Technol 101:
suggestions for improving this manuscript. We are grateful to Biosag - 5222–5228
Comércio e Serviços Agrícolas Ltda and to Msc. The authors are grateful Cao L, Jiang M, Zeng Z, Du A, Tan H, Liu Y (2008) Trichoderma
to the City Hall of Uberlândia for providing J. mimosifolia seedlings, and atroviride F6 improves phytoextraction efficiency of mustard
to João C.F. Cardoso who helped us with the PCA and the multivariate (Brassica juncea (L.) Coss. var. foliosa Bailey) in Cd, Ni contami-
linear mixed model analysis of the photosynthesis data. nated soils. Chemosphere 71:1769–1773
Cavello IA, Crespo JM, García SS, Zapiola JM, Luna MF, Cavalitto SF
Funding This study was funded by Biosag - Comércio e Serviços (2015) Plant growth promotion activity of keratinolytic fungi grow-
Agrícolas Ltda (ICIAG.PEQU.0037). CPF received a mastering course ing on a recalcitrant waste known as “hair waste”. Biotechnol Res
scholarship grant from Biosag - Comércio e Serviços Agrícolas Ltda. Int 2015:1–10. https://doi.org/10.1155/2015/952921
LCBA received a scholarship grant from Biosag. Chao Z, Yin-hua S, De-xin D et al (2019) Aspergillus niger changes the
chemical form of uranium to decrease its biotoxicity, restricts its
movement in plant and increase the growth of Syngonium
Compliance with ethical standards
podophyllum. Chemosphere 224:316–323
Chen BC, Ho PC, Juang KW (2013) Alleviation effects of magnesium on
All authors attest consent for publishing. The research project has been copper toxicity and accumulation in grapevine roots evaluated with
submitted and approved at Council of Institute of Agricultural Sciences biotic ligand models. Ecotoxicol 22:174–183
and by the Pro-Rectory of Research and Post-Graduation Studies of Chen Y, Yang W, Chao Y, Wang S, Tang YT, Qiu RL (2017) Metal-
Federal University of Uberlândia. tolerant Enterobacter sp. strain EG16 enhanced phytoremediation
using Hibiscus cannabinus via siderophore-mediated plant growth
Conflict of interest The authors declare that they have no conflict of promotion under metal contamination. Plant Soil 413:203–216
interest. Conselho Nacional do Meio Ambiente - Conama. Resolução Conama n°
420, de 28 de dezembro de 2009. Brazilian Environmental Ministry.
http://www.mma.gov.br/port/conama/res/res09/res42009.pdf.
Accessed 04 July 2019
Contreras-Cornejo HA, Macías-Rodríguez L, Cortés-Penagos C, López-
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