Interactions of Trametes versicolor, Coriolopsis rigida and the arbuscular mycorrhizal fungus Glomus deserticola on the copper tolerance of Eucalyptus globulus.

Abstract

The presence of high levels of Cu in soil decreases the shoot and root dry weights of Eucalyptus globulus. However, higher plant tolerance of Cu has been observed in the presence of the arbuscular mycorrhizal (AM) fungus Glomus deserticola. The hyphal length of G. deserticola was sensitive to low Cu concentrations, and the percentage of AM root colonisation and the metabolic activity of the AM fungus were also decreased by Cu. Therefore, a direct effect of Cu on the development of the AM fungus inside and outside the root cannot be ruled out. E. globulus colonised by G. deserticola had higher metal concentrations in the roots and shoots than do non-mycorrhizal plants; however, the absence of a higher root to shoot metal ratio in the mycorrhizal plants (1.70+/-0.11) indicated that G. deserticola did not play a filtering/sequestering role against Cu. The saprobe fungi Coriolopsis rigida and Trametes versicolor were able to remove Cu ions from the asparagine-glucose growth media. However, plants inoculated with C. rigida and T. versicolor did not accumulate more Cu than non-inoculated controls, and the growth of the plant was not increased in the presence of these fungi. However, C. rigida increased the shoot dry weight, AM root length colonisation, and metabolic mycelial activity of plants colonised with G. deserticola in the presence of Cu; only this saprobe-AM fungus combination increased the tolerance of E. globulus to Cu. Inoculation with G. deserticola and C. rigida increased the E. globulus Cu uptake to levels reached by hyperaccumulative plants.

Full text

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Technical Note
Interactions of Trametes versicolor, Coriolopsis rigida and the arbuscular
mycorrhizal fungusGlomus deserticola on the copper tolerance ofEucalyptus globulus
C. Arriagadaa,*, E. Arandab, I. Sampedrob, I. Garcia-Romerab, J.A. Ocampob
aDepartamento de Ciencias Forestales, Universidad de La Frontera, Casilla 54-D, Temuco, Chile
bEstación Experimental del Zaidín, Consejo Superior de Investigaciones Cientificas, Profesor Albareda 1, 18008 Granada-España, Spain
a r t i c l ei n f o
Article history:
Received 5 March 2009
Received in revised form 18 July 2009
Accepted 20 July 2009
Available online 18 August 2009
Keywords:
Arbuscular mycorrhizae
Heavy metals
Phytoremediation
Saprobe fungi
Soil contamination
a b s t r a c t
The presence of high levels of Cu in soil decreases the shoot and root dry weights of Eucalyptus globulus.
However, higher plant tolerance of Cu has been observed in the presence of the arbuscular mycorrhizal
(AM) fungus Glomus deserticola. The hyphal length of G. deserticola was sensitive to low Cu concentra-
tions, and the percentage of AM root colonisation and the metabolic activity of the AM fungus were also
decreased by Cu. Therefore, a direct effect of Cu on the development of the AM fungus inside and outside
the root cannot be ruled out. E. globulus colonised by G. deserticola had higher metal concentrations in the
roots and shoots than do non-mycorrhizal plants; however, the absence of a higher root to shoot metal
ratio in the mycorrhizal plants (1.70 ± 0.11) indicated that G. deserticola did not play a filtering/seques-
tering role against Cu. The saprobe fungi Coriolopsis rigida and Trametes versicolor were able to remove
Cu ions from the asparagine–glucose growth media. However, plants inoculated with C. rigida and T. ver-
sicolor did not accumulate more Cu than non-inoculated controls, and the growth of the plant was not
increased in the presence of these fungi. However, C. rigida increased the shoot dry weight, AM root
length colonisation, and metabolic mycelial activity of plants colonised with G. deserticola in the presence
of Cu; only this saprobe-AM fungus combination increased the tolerance of E. globulus to Cu. Inoculation
with G. deserticola and C. rigida increased the E. globulus Cu uptake to levels reached by hyperaccumula-
tive plants.
? 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Cu is essential for plant development and growth. However,
excessive Cu can lead to root elongation and cause damage to root
membranes. Moreover, it may cause toxicity by interfering with
photosynthesis, respiratory processes, and protein synthesis
(Marschner, 1995). White-rot fungi have to cope with toxic levels
of metal ions such as Cu often during their growth in soil.
Relatively few studies have been done using white-rot fungi in bio-
remediation of Cu-contaminated soils (Baldrian, 2003). The white-
rot fungi belonging to the Trametes and Coriolopsis genera have
been used to detoxify metal effluents from agroindustrial wastes
(Barajas-Aceves et al., 2002). These saprobe fungi are able to re-
move heavy metals such as Cu by adsorbing them on their mycelia,
and the degree of accumulation and tolerance of Cu from soil dif-
fers in different species of these fungi (Saglam et al., 1999). It is
known that white-rot fungi increase the growth of plants, espe-
cially when plants are cultivated in soils contaminated with agro-
industrial wastes (Aranda et al., 2006).
The arbuscular mycorrhizal (AM) fungi are a substantial compo-
nent of the soil microbial biomass. This symbiosis benefits plant
growth, particularly through enhanced phosphorus, water, and
mineral nutrient uptake (Smith and Read, 1997). AM fungi improve
plant resistance to the presence of high quantity of heavy metals
such as Cu in the soil. However, the effect of AM fungi on the up-
take of metals by plants is not yet totally clear. AM isolates can in-
crease or decrease metal uptake and accumulation in shoots or in
roots or can increase or reduce heavy metal translocation from
roots to shoots (Joner and Leyval, 2001; Chen et al., 2003).
Phytoremediation, the use of plants to remove toxic metals
from soils is emerging as a potential strategy for cost-effective
and environmentally friendly remediation of contaminated soils
(Glass, 2000). Some plants can accumulate high concentrations of
heavy metals and have been used in experimental assays for the
phytoremediation of contaminated soils (McGrath et al., 2002).
Many of the accumulative plants used belong to the family Brassic-
aceae; this family does not form AM symbiosis. As many hyperac-
cumulating plant families are herbaceous and non-mycorrhizal,
considerable scepticism exists about the functional importance of
AM in highly tolerant hyperaccumulating plants, but nevertheless
its functionality was recently confirmed (Regvar and Vogel-Mikus,
2008). However, these herbaceous plants produce little biomass, so
0045-6535/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2009.07.042
* Corresponding author. Tel.: +56 45 325635.
E-mail address: carriaga@ufro.cl (C. Arriagada).
Chemosphere 77 (2009) 273–278
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they are of less interest than plants with higher productivity, such
as trees (Greger and Landberg, 1999). Eucalyptus is a tree species
with a wide plasticity to grow in impoverished or marginal soils
and is able to accumulate high quantities of heavy metals (Arriag-
ada et al., 2004). Studies of AM fungal symbiosis in trees are scarce
(Wilkinson and Dickinson, 1995), but the Eucalyptus species were
able to develop AM symbiosis (Arriagada et al., 2004). To our
knowledge, there are few reports on the effect of AM fungi on Cu
phytoextraction by high-biomass crops such as maize and Eucalyp-
tus from Cu-contaminated soils (Arriagada et al., 2004; Wang et al.,
2007). Cultivation of non-hyperaccumulating, highly mycorrhizal
plants that produce large amounts of biomass, such as Eucalyptus
and Populus trees, on contaminated soil is recommended as a phy-
toremediation practice to prevent food chain contamination be-
cause of their capacity to accumulate heavy metals in the stem-
wood (Leep and Dickinson, 1998; Arriagada et al., 2004; Komarek
et al., 2008; Lingua et al., 2008).
In addition, it is known that soil microorganisms such as sap-
robe fungi affect AM symbiosis. Some experimental results confirm
the existence of synergistic, neutral, and antagonistic effects of
saprobe fungi on plant root colonisation by AM fungi (Fracchia
et al., 1998).
The aim of this work is to determine if the interaction between
AM and saprobe fungi increases the tolerance of Eucalyptus to high
concentrations of Cu in soil.
2. Materials and methods
2.1. Arbuscular mycorrhizal (AM) fungi
The AM Glomus deserticola (Trappe, Bloss and Menge) from the
Instituto de Investigaciones Agrobiológicas de Galicia was used.
2.2. Saprobe fungi
The saprobe fungiCoriolopsis rigida andTrametes versicolor were
isolated by the particle washing method using a multichamber
washing apparatus (Widden and Bisset, 1972). These fungi were
classified as described by McAllister (1992). Strains are kept at
the fungal culture collection of the Facultad de Ciencias Agropecua-
rias y Forestales, Universidad de La Frontera in Temuco, Chile. Both
saprobe fungi were transferred to tubes of 39 g L?1potato dextrose
agar (PDA, DIFCO) and 2% malt extract at 4 ?C as stock culture.
2.3. In vitro experiments
The effect of Cu on spore germination and hyphal length of G.
deserticola was tested in 9-cm diameter plastic Petri dishes. The
sporesofG.deserticolaweresurface-sterilisedasdescribedbyMosse
(1962). Ten surface-sterilised spores per plate were placed 1 cm
from the edge of a Petri dish with 10 mL of 10 mM 2-(N-morpholin)
ethane sulphonic acid buffer (pH 7) plus 0.04 g of Gel-Gro (ICN Bio-
chemicals, Aurora, OH, USA). The quantity of 39.6, 79.2, 118.8,
237.6and396.5 mgofCuSO4?5H2OwereaddedtoPetridishesbefore
thesolidificationmediumtoreachafinalconcentrationsof0,10,20,
30, 60, and 100 mg Cu L?1. The plates were incubated at 25 ?C in the
dark for 21 d and were sealed to reduce dehydratation and contam-
ination. Hyphal length of the germinated G. deserticola spores was
determined under a binocular stereo microscope (Olympus SZ-PT)
at40?magnificationattheendoftheexperimentusingthe gridline
intersect method (Marsh, 1971). Ten replicates petri dishes with 10
spores each were used and all the fungal mycelia were measured.
An aqueous suspension in sterile distilled water containing
mycelium of the saprobe fungi was prepared from cultures grown
in PDA for 1 week at 27 ?C. Two ml of this suspension were added
to 250-mL flasks containing 125 mL of sterile AG liquid medium
(Galvagno, 1976) in a shaker at 28 ?C. The AG medium consisted
of 1 g glucose, 0.4 g asparagine, 0.05 g MgSO4, 0.05 g KH2PO4and
100 mL distilled water. The quantity of 0.039, 0.39, 1.98 and
3.96 g of CuSO4?5H2O were added to AG medium to reach a final
concentration of 0, 10, 100, 500, and 1000 mg Cu L?1. The concen-
tration of Cu was analysed in the AG medium after 2 week culture
of C. rigida and T. versicolor by atomic absorption spectroscopy
(Perkin-Elmer 5380, Norwalk, CT, USA) after microwave digestion
with a mixture of H2SO4and H2O2(Mingorance, 2002). Ten repli-
cates were used in these experiments.
2.4. Greenhouse experiments
The experiments were carried out using Eucalyptus (Eucalyptus
globulus Labill) as test plants. Seeds were surface-sterilised with
HgCl2for 10 min and thoroughly rinsed with sterilised water and
sowninmoistenedsand. Aftergermination,uniformseedlingswere
planted in 0.3-L pots (One seedling per pot), filled with a mixture of
sterilised sand:soil at a proportion of 1:1 (v:v). The soil, classified as
an Andisol (Acrudoxic Hapludands), is moderately acidic (pH 5.5)
with good drainage and water infiltration. Plants were grown in a
greenhouse with supplementary light provided by Sylvania incan-
descent and cool-white lamps, 400 E m?2s?1, 400–700 nm, with a
16/8 h day/nightcycle at 25/19 ?C and 50% relative humidity. Plants
were watered from below and fertilised every week with 10 mL of a
nutrient solution plus 50 mg L?1of P (Hewitt, 1966). The AM fungal
inoculum was a root-and-soil inoculum consisting of rhizosphere
soil containing spores (approximately 1000 spores per 100 g?1)
and colonised root fragments of Medicago sativa. The inoculation
plants were in amounts of 8 g of soil inoculum per pot, an amount
that was predetermined to enable high levels of root colonisation.
In order to restore the microbial population present in the soil inoc-
ulum, uninoculated plants were given a filtrate (Whatman No. 1 pa-
per) of the inoculum containing common soil microflora that was
free of AM fungal propagules.
We inoculated E. globulus pots with: (1) G. deserticola, (2) C. rig-
ida or T. versicolor, and (3) C. rigida or T. versicolor plus G. deserticola,
as well as we kept some seedlings as controls. Plants were inocu-
lated at the time of transplanting (after 3 week of growth). The sap-
robe fungi were inoculated at the same time as was G. deserticola.
The quantity of 0.039, 0.39, 1.98, 3.96 and 7.93 g of CuSO4?5H2O
were applied to E. globulus pots to reach the concentrations of 0,
10, 100, 1000, up to 2000 mg Cu kg?1of sand:soil. Ten replicate
pots per treatment and Cu concentration were used.
Plants were harvested after 12 week and dry mass was deter-
mined. After the harvest, two samples of fresh weight were taken
from the entire root system at random. One of the samples was
cleared and stained (Phillips and Hayman, 1970), and the percent-
age of root length colonisation with AM fungus was measured by
the gridline intersect method (Giovannetti and Mosse, 1980). In
the other sample, succinate dehydrogenase (EC 1.3.99.1) (SDH)
activity was measured in fungal mycelia by the reduction of tetra-
zolium salts (Natrium blue tetrazolium from Sigma Chemicals) at
the expense of added succinate (Succinic acid disodium salt from
Fluka Analytical), (MacDonald and Lewis, 1978); the percentage
of AM fungal mycelia with SDH activity was determined under a
compound microscope (Ocampo and Barea, 1985).
We measured the following response variables; total Cu content
in the root and shoot of 10 E. globulus seedlings per treatment. Cu
concentrations were measured by atomic absorption spectroscopy
(Perkin-Elmer 5380, Norwalk, CT, USA) after microwave digestion
with a mixture of H2SO4and H2O2according to the procedure of
Mingorance (2002).
We studied the following three main factors and their respec-
tive levels as follows AM fungal (control and G. deserticola), Sap-
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robe fungi (control, C. rigida and T. versicolor), and Cu supply (in
sand:soil pot at 0, 10, 100, 1000 and 2000 mg Cu kg?1). We also
analysed the interaction among the main factors using a factorial
analysis of variance. Statistical analyses were conducted in SPSS
software, version 11.0 (SPSS Inc., 1989–2001).
3. Results
The length of AM fungal mycelia was strongly reduced by Cu
presence, even at the lowest concentration (10 mg L?1) to a
5 mm (Fig. 1). The concentration of 1000–10 mg mL?1Cu in the
AG growth medium decreased between 53% and 66% after culture
of C. rigida and between 26% and 47% after culture of T. versicolor.
Factorial analyses results were as follows. We found significant
differences in the population means of all the response variables
(shoot dry weight, Cu in shoot, and Cu in root) to the main factors
AM fungal (P < 0.01) and Cu supply (P < 0.001), but not to the factor
saprobe fungi. We conducted contrasts between a main factor and
levels of some factors in order to assess their interactions. The fol-
lowing contrasts between; the entire factors AM fungal and Cu
supply, C. rigida and the factor AM fungal, as well as between T. ver-
sicolor and the factor AM fungal, were found statistically significant
(P < 0.01, and P < 0.05 for the last ones, respectively) for all the re-
sponse variables. Nevertheless, we did not find significant interac-
tions between any of the levels of saprobe fungi (C. rigida and T.
versicolor) and the factor Cu supply. Finally, we further explore
the interaction of T. versicolor and C. rigida by themselves with
the factors AM fungal and Cu supply. Both saprobe fungi shown
significant interactions with those factors (P < 0.05), although T.
versicolor shown a stronger difference in the population mean of
the variable Cu in root (P < 0.01).
The shoot dry weight average for each factor and their interac-
tions in Fig. 2a illustrate that saprobe fungi did not provide an
additional Cu tolerance to E. globulus. However, the mycorrhizal
fungus G. deserticola inoculated alone increased the shoot dry
weight of E. globulus such that it was significantly higher, even at
0
5
10
15
20
25
0 10
Cu concentration (mg L¯¹ )
2030 60 100
Hyphal length (mm)
Fig. 1. Effect of Cu on the hyphal length Glomus deserticola spores. The data are
shown as mean ± standard error of the mean (n = 10).
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Shoot dry weight (g)
010100 10002000
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Control Glomus
deserticola
Trametes
versicolor
Coriolopsis
rigida
G.deserticola
+T.versicolor
G.deserticola
+ C.rigida
Root dry weight (g)
a
b
Fig. 2. Shoot (a) and root (b) dry weight of Eucalyptus globulus inoculated or not with AM or with the saprobe fungi in soil contaminated with different Cu concentrations (0,
10, 100, 1000 and 2000 mg Cu kg?1sand:soil). The data are shown as mean ± standard error of the mean (n = 10).
C. Arriagada et al./Chemosphere 77 (2009) 273–278
275

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the application rate of 100 mg Cu kg?1sand:soil. The application of
doses higher than 100 mg Cu kg?1sand:soil decreased the shoot
dry weight of plants in all treatments tested, except when C. rigida
was inoculated together with G. deserticola, which decreased only
at 2000 mg Cu kg?1sand:soil. In our experiments, E. globulus did
not show any visible sign or symptom of copper toxicity from each
treatment.
The root dry weight means for each factor and their interac-
tions, illustrated in Fig. 2b, show that the root dry weight of plants
inoculated with G. deserticola in either the absence or presence of
all Cu doses was higher than the non-AM-inoculated plants. Inoc-
ulation with C. rigida or T. versicolor did not increase the root dry
weight of plants colonised by G. deserticola (Fig. 2b).
The effect of Cu treatment on mycorrhizal root colonisation and
SDH activity of E. globulus decreased in the presence of
100 mg Cu kg?1sand:soil (Fig. 3a and 3b). Plants inoculated with
T. versicolor did not have increased AM root colonisation or SDH
activity of E. globulus colonised by G. deserticola under any treat-
ment. However, at all Cu doses, the percentage of root colonisation
and the SDH activity of G. deserticola were both higher in the pres-
ence than in the absence of C. rigida.
The Cu concentration in shoots and roots of E. globulus plants
did not show any differences at 10 and 100 mg Cu kg?1sand:soil,
and in plants growing at such concentrations, the shoot and root
concentrations of Cu ere not affected either by AM fungi or by sap-
robe
2000 mg Cu kg?1sand:soil, G. deserticola produced a significant in-
crease in shoot and root Cu concentration; this increase was only
reinforced by the inoculation of G. deserticola together with C. rig-
ida at 2000 mg Cu kg?1sand:soil.
Root to shoot metal content ratios of E. globulus were not af-
fected by the presence of either AM (1.70 ± 0.11), saprobe fungi
(1.69 ± 0.15) or non inoculated controls (1.75 ± 0.09) and non sig-
nificant differences between all treatments were observed (data
not shown).
fungi inoculation(Fig.4).However, at 1000 and
4. Discussion
The growth of E. globulus, like other plants, was affected by the
presence of a high level of Cu in the soil (Marschner, 1995). Cu con-
centrations inhibiting shoot dry weight were higher in AM than in
non-AM E. globulus plants, thus indicating higher plant tolerance to
Cu soil contamination as a result of AM colonisation. Colonisations
by AM fungi are characterised by uptake of Cu to the colonised
plant. However, in contrast with other essential metals, Cu is toxic
to most fungi even at very low concentrations (Baldrian, 2003). The
hyphal length of the AM fungus G. deserticola was sensitive to low
concentrations of Cu, indicating that this metal will affect the
development of this fungus outside the root. Cu also decreased
the percentage of AM root length colonisation and the metabolic
0
10
20
30
40
50
60
70
80
Glomus deserticola G.deserticola + T.versicolorG.deserticola + C.rigida
SDH Activity (%)
10
20
30
40
50
60
70
80
Mycorrhizal colonization (%)
0 10100 10002000
a
b
Fig. 3. Effect of AM and saprobe fungi on root length colonisation (a) and percentage of AM mycelium with SDH activity (b) of Eucalyptus globulus seedling in soil with
different Cu concentrations (0, 10, 100, 1000 and 2000 mg Cu kg?1sand:soil). The data are shown as mean ± standard error of the mean (n = 10).
276
C. Arriagada et al./Chemosphere 77 (2009) 273–278

Page 5

activity of the arbuscular fungi, measured as SDH activity of the
fungal mycelium inside the root of E. globulus. It is possible that
the negative effect of Cu on AM symbiosis can be due partly to
the toxic effect of Cu on the plants and partly to Cu inhibition of
the extraradical development of the AM fungi. Nevertheless, due
to the decrease in the metabolic activity of the AM fungi, a direct
effect of Cu on the development of the AM fungi inside the root
cannot be discarded. However, AM fungi increased the plant Cu
concentration when grown in soil with a high concentration of this
metal (Carvalho et al., 2006). In fact, we found that E. globulus col-
onised by G. deserticola in the presence of high Cu levels had higher
metal concentrations in the roots and shoots than non-mycorrhizal
plants. Mycorrhizal plants have various heavy metal detoxification
mechanisms, including the retention of toxic metals in roots and
the subsequent reduction of translocation to shoots (Hildebrandt
et al., 2007). However, the absence of a higher root to shoot metal
ratio in E. globulus mycorrhizal plants (1.70 ± 0.11) did not support
the hypothesis that the AM fungus G. deserticola plays a filtering/
sequestering role against Cu. At present, it is not known what hap-
pens to Cu taken up by mycorrhizae and whether this metal could
be transformed into unknown nontoxic compounds in E. globulus
(Orłowska, et al., 2008). Plants have their own protection mecha-
nisms against metal toxicity. It is known that E. globulus was able
to accumulate heavy metals in the stem more than in the leaves,
and the AM fungi seem to contribute to this redistribution of some
heavy metals inside the E. globulus plant (Arriagada et al., 2004).
This process decreased the damage caused to the physiology of
the plant more than when the heavy metals were accumulated in
the metabolically active parts of the plant (Leep and Dickinson,
1998).
The white-rot fungi C. rigida and T. versicolor were able to re-
move Cu ions from the growth media, possibly by adsorbing them
on their mycelia (Jarosz-Wilkolazka et al., 2002; Bayramoglu et al.,
2003). However, plants inoculated with C. rigida and T. versicolor
did not accumulate more Cu than the noninoculated controls,
and the growth of the plant was not increased in the presence of
these fungi. These results indicate that the effect of the white-rot
fungi on the tolerance of E. globulus to Cu was mediated by its ef-
fect on the colonisation and metabolic activity of the AM fungi. In
our experiments, C. rigida, but not T. versicolor increased the shoot
dry weight, as well as the root length colonisation and metabolic
activity of the AM fungus of plants colonised by G. deserticola in
presence of Cu. Only the C. rigida-G. deserticola combination in-
creased the tolerance of E. globulus to Cu. It is known that there
is synergistic action of some soil saprobe fungi on the AM colonisa-
tion of root and on the tolerance of AM plants to heavy metals
(Fracchia et al., 1998; Arriagada et al., 2004).
The concentration of Cu in the shoot must be higher than
1000 mg kg?1to be considered hyperaccumulative plant (Regvar
and Vogel-Mikus, 2008). The Cu uptake level reached by E. globulus
was lower than 1000 mg kg?1, indicating that this plant cannot be
considered a hyperaccumulative plant of this heavy metal. In the
present study, E. globulus did not show any visible sign or symptom
of copper toxicity and the higher root concentrations of Cu were
0
100
200
300
400
500
600
700
800
900
1000
Cu in Shoot (mg kg¯¹ dry mass)
0 10100 10002000
100
200
300
400
500
600
700
800
900
1000
Control Glomus
deserticola
Trametes
versicolor
Coriolopsis
rigida
G.deserticola
+ T.versicolor
G.deserticola
+ C.rigida
Cu in Root (mg kg¯¹ dry mass)
Fig. 4. Cu concentration in shoot and root of Eucalyptus globulus inoculated or not with AM or with the saprobe fungi in soil with different Cu concentrations (0, 10, 100, 1000
and 2000 mg Cu kg?1sand:soil). The data are shown as mean ± standard error of the mean (n = 10).
C. Arriagada et al./Chemosphere 77 (2009) 273–278
277