How mycorrhizal fungi reduce copper toxicity in tropical trees

Uptake and translocation of copper by mycorrhized seedlings
Sterculia setigera (Del.) under Copper-contamined soil
Keywords:
Arbuscular mycorrhiza, heavy metals, Sterculia setigera, uptake,
translocation.
ABSTRACT:
Pot culture experiments were established to determine the effects of
arbuscular mycorrhizal fungus (AMF) (Glomus fasciculatum) on tropical gum tree
(Sterculia setigera Del.) grown in Copper contaminated soils. AMF and non-AMF
inoculated plants were grown in sterilized substrates and subjected to different
copper level (0, 200, 400,600, 800 mg kg-1
) concentrations. Root and shoot biomasses
of inoculated plants were significantly higher than those of non-inoculated. Copper
concentrations in roots were significantly higher than those in shoots in both the
inoculated and non-inoculated plants, indicating this heavy metal mostly accumulated
in the roots of plants. Copper translocation efficiency from root to shoot was lower in
mycorrhizal plants than in nonmycorrhizal ones at any Copper addition levels.
However, at high soil Copper concentrations, shoot Copper concentration of
inoculated plant were significantly reduced by about 50% compared to non-
inoculated plants. These results indicated that AMF could promote tropical gum tree
growth and decrease the uptake of Cu at higher soil concentrations, thus protecting
their hosts from the toxicity of Copper contaminated soils.
022-028 | JRA | 2012 | Vol 1 | No 1
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www.jagri.info
Journal of Research in
Agriculture
An International Scientific
Research Journal
Authors:
Malick Ndiaye1
, Cavalli
Eric2
, Diouf Adama1
, Diop
Tahir Abdoulaye1
.
Institution:
1. Laboratoire de
Biotechnologies des
Champignons, Département
de Biologie Végétale,
Faculté des Sciences et
Techniques, Université
Cheikh Anta Diop, BP. 5005
Dakar-Fann, Sénégal.
2. Service d’Analyse et de
Caractérisation, UFR
Sciences et Techniques,
Université de Franche-
Comté, 16, route de Gray -
25030 Besançon cedex,
France.
Corresponding author:
Malick Ndiaye
Email:
papmalic@yahoo.fr,
eric.cavalli@univ-fcomte.fr,
adiouf97@yahoo.com,
diopta@yahoo.fr,
Tél/Fax :
(221) 33 864 6658
Phone:
+ 221 77 534 84 79,
33 614 93 47 38,
+ 221 77 596 77 17,
+ 221 77 630 59 57.
Web Address:
http://www.jagri.info
documents/AG0012.pdf.
Dates:
Received: 14 Dec 2011 /Accepted: 25 Dec 2011 /Published: 24 Jan 2012
Article Citation:
Malick Ndiaye, Cavalli Eric, Diouf Adama, Diop Tahir Abdoulaye.
Uptake and translocation of copper by mycorrhized seedlings Sterculia setigera (Del.)
under Copper-contamined soil.
Journal of Research in Agriculture (2012) 1: 022-028
Original Research
Journal of Research in Agriculture
JournalofResearchinAgriculture An International Scientific Research Journal

INTRODUCTION
Toxic metal accumulation in soils of
agricultural interest is a serious problem needing
more attention and investigations on soil-plant
metal transfer must be pursued to better understand
the processes involved in metal uptake. Even at low
concentrations in the environment, excessive levels
of Copper are not only toxic to plants, but also to
humans through the food chain and pose a potential
threat to human health, environmental quality and
sustainable food production (Tao et al, 2003; Menti
et al, 2006). To prevent such risks, it is necessary to
quantify Copper transfer to plants. Several studies
investigated soil-plant metal transfer to find the
most reliable methods for the prediction of heavy
metal bioavailability and to understand the
processes involved in the uptake of these toxic
elements. In recent years, several studies have
shown that some plants are capable of absorbing
and / or transferring the metal in the roots of plants
(Song et al, 2004, Leung et al, 2007).
Arbuscular mycorrhizal (AM) fungi increase
nutrient acquisition by exploring a vast soil volume
(Smith and Read, 2008) and can be beneficial to
host plant growing in unfavorable soil conditions as
in nutrient deficient soils or in polluted areas. AMF
are known to influence metal transfer in plants by
increasing plant biomass and reducing metal
toxicity to plants, even if diverging results were
reported (Heggo et al, 1990 Leyval et al, 1997;
Ouziad et al, 2005; Requena, 2005; Hildebrandt et
al, 2007). In addition, fungi also affect the AM
uptake of metals by plants, soil and transfer of the
root. The alleviation of stress may be partly due to
immobilization of heavy metals in
mycorrhizosphere and to the decrease of metal
concentration in mycorrhizal plants (Kapoor and
Bhatnagar, 2007). On the other hand, under
conditions of high available soil Copper
concentrations of this trace element in shoots have
been reported to be lower in mycorrhizal than in
non-mycorrhizal plants (Weissenhorn et al, 1995;
Liu et al, 2000).
The aim of this study was thus to assess the
uptake and phytotoxicity of Cu in tropical gum tree
(Sterculia setigera) in Copper-contaminated soils.
MATERIALS AND METHODS
Soil
Soil used in this study was collected at 5-20
cm depth from soil botanical garden department at
latitude 14°41’2’’N, longitude 17°27’45’’W
(Cheikh Anta Diop University /Senegal). Soil
characteristics following: clay 3.6%, silt 1.6%, fine
silt 2.9%, fine sand 51%, coarse sand 40.9%,
organic matter 1.06%, total C 2.5, total N 0.33, total
P 47µg.g-1
, available P 3.1µg.g-1
, pH (sol/water
ratio 1:2) 6.7, pH (sol/KCL ratio 1:2) 4.5, was taken
for the analysis.
Mycorrhizal inoculum
Mycorrhizal inoculums containing
indigenous species G. fasciculatum was obtained
from Laboratory of Fungal Biotechnology (LBC) of
the department of Plant Biology (Cheikh Anta Diop
Universty / Senegal) was multiplied by using maize
as host plant. Mycorrhizal inoculum consited of
rhizospheric soil mixture from pure culture
containing spores, hyphae and mycorrhizal root
fragments (an average of 40 spores per gram and
85% of roots infected) were used for the
experiment.
Experimental procedure
Soil was sterilized by autoclaving at 120°C,
for 1h. Experiment was laid in a randomized block
with five replicates. Two factors were studied: (a)
Copper addition levels and (b) inoculation. Seeds of
S. setigera were scarified by the addition of sulfuric
acid (H2SO4 96%) for 100 min, and rinsed in sterile
distilled water. After successive five min baths in
sterile distilled water, seeds were germinated in
jars. The jars previously sterilized by autoclaving at
120°C for 20 min and were cotton soaked.
Germination occurs in the dark in an oven at 32°C
for 3 days. Two seedlings of S. setigera were
transferred to bags used in nursery and one seedling
was left after emergence.
Five Copper addition levels (0, 200, 400,
600 and 800 mg kg-1
) were applied in an analytical
grade CuSO4 solution mixed thoroughly with soil.
During this procedure, plants were incoulated with
AM fungus G. fasciculatum by placing 20 g of
inoculum directly in the substrate at the position of
the roots (the control without AM fungal
propagules).
Plants were grown in greenhouse with
following conditions: day/night cycle of 12/12h,
32/25°C and 40-50% air humidity. Plants were
watered with tap water.
Plant and soil analyses
To determine the degree of colonization
after 12 week culture, a portion of the roots (about
1 g fresh weight) was washed with tap water, and
then fully rinsed in distilled water. The clean roots
were cut into segments around 1 cm long, cleared
by soaking in 10% KOH and stained according to
Phillips and Hayman, (1970). Percentage
023 Journal of Research in Agriculture (2011) 1: 022-028
Ndiaye et al.,2011

colonization was determined by the grid intersect
method followed by Giovannetti and Mosse,
(1980). Root and shoot dry weights were measured
after oven-drying at 70 °C for 48h.
Chemical analyses were done at the Water
Chemistry Laboratory of Analysis and
Characterization Service (SERAC) of the
University of Franche-Comte of France according
to standardised French procedures (AFNOR).
Copper concentrations in dried and ground plant
material were determined by Inductively Coupled
Plasma - Optical Emission Spectrometry (ICP-
OES) after wet-digestion with a mixture of
concentrated HNO3 and HClO4 (3:2, v/v,
guaranteed reagent) mixed acid. pH was
determined in a 1:2.5 (w/v) soil/water suspension.
Three aspects of plant Copper efficiency were
assessed. According to Harper et al. (1997), Cu
uptake efficiency was calculated based on the
ability of the root to take up Copper from the soil
(the total amount of Copper in the plant expressed g
-1
root dry weight) and the Copper translocation
ability was computed as the ability of the plant to
transport the Copper to the shoot (percentage of
total Cu in the plant present in the shoot tissue):
Statistical analysis
Statistical procedures were carried out with the
software package R version 2.5. Two factor
analyses of variance (ANOVA) were performed to
partition the variance into the main effects and the
interaction between Inoculation and Cu addition
level.
RESULTS
Plant Biomass
No differences of shoot biomass were found
at 0 and 200 mg kg-1
Copper. However, a decrease
was observed in shoot biomass at 400 and 600 mg
kg-1
Copper (Fig. 1). Root dry weights showed a
similar trend. At 600 mg kg-1
Copper, both shoot
and root dry weights were decreased. Compared
with controls, AMF inoculation increased shoot and
root dry weights at any Copper levels (Fig. 1a, 1b).
Copper concentrations
In general, both shoot and root Copper
concentrations tended to increase with increasing
Copper addition levels. Compared with control
plants, shoot Cu concentrations in mycorrhizal
plants were higher with no Cu addition but lower at
other levels. Root Cu concentrations of inoculated
plant were higher even 400 mg kg-1
Cu added (Fig.
2). At 600 mg kg-1
Cu added, no significant
difference was found between root inoculated and
non inoculated plants. Both inoculation and Cu
addition had a significant effect on shoot and root
Cu concentrations, and the interactions between
them were also significant for shoot and root Cu
concentrations (Table 1).
Cu uptake
On the whole, shoot Cu uptake increased
with more Cu added while shoot Cu uptake did not
change consistently with Cu added for both non-
mycorrhizal and mycorrhizal treatments (Fig. 3).
Shoot Copper uptake in mycorrhizal plants was
significantly higher compared to non-mycorrhizal
control on zero Cu addition but lower at other levels
(Fig. 3a). Copper uptake did not change
Journal of Research in Agriculture (2011) 1: 022-028 024
Ndiaye et al.,2011
Cu addition levels(mg kg1
)
Figure 1: Shoot (a) and root (b) dry weights of S.
setigera plants under different treatments. C and T
represent non inoculated and inoculated with
mycorrhizal fungus G. fasciculatum. Vertical bars
represent mean standard errors (S.E.).
Cu addition levels(mg kg1
)
Root dry weight
Cu uptake of the plantsUptake efficiency
(µg g-1
) =
Translocation
efficiency =
Shoot Cu
Root Cu
Phytoextraction
efficiency (µg g-1
) =
Root dryweight
Shoot Cu uptake

significantly with 200 mg kg-1
Copper added,
increased with zero, 200 and 400 mg kg-1
Cu (Fig.
3b). Both inoculation and Copper addition had a
significant effect on shoot and root Copper uptake,
and the interactions between them were also
significant for shoot and root Copper uptake (Table
1).
Cu uptake efficiency, translocation efficiency
and phytoextraction efficiency
We calculated total uptake of Copper
removed through harvesting each part by
multiplying the biomass per pot by the average
metal concentrations in different plant Copper
uptake efficiency and phytoextraction efficiency
both increased with increasing amounts of Copper
added, while translocation efficiency showed the
opposite trend (Fig. 4). Copper uptake efficiency,
phytoextraction efficiency and Copper translocation
efficiency was lower in mycorrhizal plants than in
non-mycorrhizal ones at any Copper addition levels
(Fig. 4a, 4b and 4c).
DISCUSSION
Heavy metal stress significantly reduced
shoot and root dry matter compared with the control
treatment. However, AM colonization significantly
improved these parameters in the heavy metal-
stressed plants but they remained lower than the
values for control plants in all cases. AM
colonization also significantly improved shoot and
root dry matter, but it did not significantly affect
root dry matter in control plants. Mycorrhizal
colonization decreased Copper concentrations in the
roots and shoots under 3.5 and 100 mg kg-1
Copper
treatments, with a concomitant increase in root and
shoots biomass. It is possible that the reduced
Copper concentrations were partially due to the
improved growth as a result of mycorrhizal
colonization. Mycorrhizal plants may release more
root exudates containing soil enzymes than that of
non-mycorrhizal plants because of the larger root
system and/or improved nutrition and/or resistances
to stress of mycorrhizal plants (Rao and Tak, 2001).
Ndiaye et al.,2011
Variables Inoculation
Cu
addition levels
Inoculation x Cu
addition levels
Df 1 3 3
Shoot Cu
concentration
1775.74*** 10186.34*** 272.98***
Root Cu
concentration
1184789*** 461382*** 310863***
Shoot Cu
uptake
54827.4*** 34163.3*** 6445.8***
Root Cu
uptake
1547664*** 1294972*** 361220***
Table 1: Significance level (F-values) of effects of differents factors and factors interaction
on variables based on analysis of variance (ANOVA).
Figure 2: Shoot (a) and root (b) Cu concentrations of Sterculia setigera plants under different treatments. C and
T represent noninoculation and inoculation with mycorrhizal fungus G. fasciculatum. Vertical bars represent
mean standard errors (S.E.).

Relative higher yielding plants decreases the
concentrations of heavy metals in plants, especially
in shoots, (Gonzalez-Chavez et al, 2002). On the
other hand, it was also observed that AMF
inoculation either increased heavy metal content in
plants, leading to an inhibitory effect on plant
biomass (Gildon and Tinker, 1983; Weissenhorn
and Leyval, 1995). Sometimes, AMF inoculation
improved metal concentrations and plant biomass
(Davies et al, 2002). It has been well documented
that mycorrhizal colonization can have significant
impacts on metal uptake by host plants
(Weissenhorn and Leyval, 1995; Chen et al, 2007).
The mechanisms to explain the altered uptake have
mainly focused on metal immobilization in the root
and/or mycorrhizosphere (Marschner, 1995).
The results of the current experiments
showed that mycorrhizal colonization improves the
ability of Sterculia setigera plants to resist Copper
toxicity. Mycorrhizal colonization decreased
Copper concentrations in the roots and shoots under
400 and 600 mg.Kg-1
Cu treatments. It is possible
that the reduced Copper concentrations were
partially due to the improved growth as a result of
mycorrhizal colonization, i.e. growth dilution
effect, but the results presented here suggested that
Copper immobilization on roots could be a major
factor. Extensive binding of Cu to roots and
mycorrhizae has been shown in other studies
(Turnau, 1998; Kaldorf et al, 1999). AMF were also
shown to confer enhanced resistance (Gonzalez-
Chaves et al, 2002). In line with all these findings,
Ndiaye et al.,2011
Figure 4: Cu uptake efficiency (a), phytoextraction efficiency (b) and translocation efficiency (c) of Sterculiasetigera plants
under different treatment. C and T represent noninoculation and inoculation with mycorrhizal fungus G. fasciculatum.
Figure 3: Shoot (a) and root (b) Cu uptake of Sterculia setigera plants under different treatments. C and T
represent noninoculation and inoculation with mycorrhizal fungus G. fasciculatum. Vertical bars represent
mean standard errors (S.E.).
RootCuuptake(mgkg1
)
ShootCuuptake(mgg-1
)

AMF colonized plants grown in heavy metal
contaminated soil, and also from another location,
was found to contain lower levels of heavy metals
in roots and shoots than the non-colonized control
plants (Kaldorf et al, 1999).
These results indicate the positive impact of
G. fasciculatum in enhancing not only Copper
uptake in Sterculia plants but also root to shoot
translocation of Copper. It also indicates that
Copper concentration in shoot of Sterculia can be
modulated by AM fungi when growing in soil
contaminated with Copper.
CONCLUSION
The current study was a short-term
greenhouse study that indicated the beneficial role
of AM fungi in enhancing plant growth, Copper
uptake by root and root to shoot Copper
translocation. However, longer-term verification of
the results is necessary. The mechanisms
responsible for increased Copper uptake and
translocation in Sterculia are still unclear.
Moreover, uptake of Copper is strongly influenced
by AMF. Further studies are required to test the
efficacy of AM fungi in enhancing metal uptake in
soils with varying AMF.
ACKNOWLEDGEMENTS
The authors wish to thank to Asyila Gum
Company and Analysis and Characterization
Service (SERAC) at University of Franche-Comté
in France.
REFERENCES
Chen BD, Zhu YG, Duan J, Xiao XY and Smith
SE. 2007. Effects of the arbuscular mycorrhizal
fungus Glomus mosseae on growth and metal
uptake by four plant species in copper mine tailings.
Environ. Pollut., 147:374-380.
Davies Jr FT, Puryear JD, Newton RJ, Egilla JN
and Grossi JAS. 2002. Mycorrhizal fungi increase
chromium uptake by sunflower plants: Influence on
tissue mineral concentration, growth, and gas
exchange. J. Plant Nutr., 25:2389-2407.
Giovannetti M, Mosse B. 1980. An evaluation of
techniques for measuring vesicular-arbuscular
mycorrhizal infection in roots. New Phytol., 84:489-
500.
Gonzalez-Chavez C, D’Haen J, Vangronsveld J
and Dodd JC. 2002. Copper sorption and
accumulation by the extraradical mycelium
ofdifferent Glomus spp. (arbuscular mycorrhizal
fungi) isolated from the same polluted soil. Plant
and Soil., 240:287-297.
Harper FA, Smith S and Macnair M. 1997. Can
an increased copper requirement in copper-tolerant
Mimulus guttatus explain the cost of tolerance? I.
Vegetative growth. New Phytol., 136:455-467.
Heggo A, Angle JS and Chaney RL. 1990. Effects
of vesicular-arbuscular mycorrhizal fungi on heavy
metal uptake by soybeans. Soil Biology and
Biochemistry 22:865-869.
Kaldorf M, Kuhn AJ, Schroder WH,
Hildebrandt U and Bothe H. 1999. Selective
element deposits in maize colonized by a heavy
metal tolerance conferring arbuscular mycorrhizal
fungus. J. Plant Physiol., 154:718-728.
Kapoor R, Bhatnagar AK. 2007. Attenuation of
cadmium toxicity in mycorrhizal celery (Apium
graveolens L.). World Journal of Microbiology and
Biotechnology 23:1083-1089.
Leung HM, Ye ZH and Wong MH. 2007.
Survival strategies of plants associated with
arbuscular mycorrhizal fungi on toxic mine tailings.
Chemosphere 66:905-915.
Leyval C, Turnau K and Haselwandter K. 1997.
Effect of heavy metal pollution on mycorrhizal
colonization and function: physiological, ecological
and applied aspects. Mycorrhiza 7:139-153.
Liu A, Hamel C, Hamilton RI, Ma B and Smith
DL. 2000. Acquisition of Cu, Zn, Mn and Fe by
mycorrhizal maize (Zea mays L.) grown in soil at
different P and micronutrient. Mycorrhiza 9:331-
336.
Marschner H. 1995. Mineral nutrition of higher
plants. Academic Press, London.
Menti J, Roulia M, Tsadilas E and
Christodoulakis NS. 2006. Longterm application
of sludge and water from a sewage treatment plant
and the aftermath on the almond trees (Prunus
dulcis). Bulletin of Environmental Contamination
and Toxicology 76(6):1021-1030.
Ndiaye et al.,2011

Ouziad F, Hildebrandt U, Schmelzer E and
Bothe H. 2005. Differential gene expressions in
arbuscular mycorrhizal-colonized tomato grown
under heavy metal stress. Journal of Plant
Physiology 162:634-649.
Phillips JM, Hayman DS. 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.
Requena N. 2005. Measuring quality of service:
phosphate “à la carte” by arbuscular mycorrhizal
fungi. New Phytologist 168:268-271.
Smith SS, Read DJ. 2008. Mycorrhizal symbiosis.
Cambridge, UK: Academic Press.
Song J, Zhao FJ, Luo YM, Mc Grath, SP and
Zhang H. 2004. Copper uptake by Elsholtzia
splendens and Silene vulgaris and assessment of
copper phytoavailability in contaminated soils.
Environ Pollut., 128:307-315.
Tao S, Chen YJ, Xu FL, Cao J and Li BG. 2003.
Changes of copper speciation in maize rhizosphere
soil. Environmental Pollution 122(3):447-454.
Turnau K. 1998. Heavy metal content and
localization in mycorrhizal Euphorbia cyparissias
from zinc wastes in southern Poland. Acta Soc. Bot.
Pol., 67:105-113.
Weissenhorn I, Leyval C. 1995. Root colonization
of maize by a Cd-sensitive and a Cd-tolerant
Glomus mosseae and cadmium uptake in sand
culture. Plant Soil., 175:233-238.
Ndiaye et al.,2011
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