Uptake and translocation of copper by mycorrhized seedlings Sterculia setigera (Del.) under Copper - contamined soil

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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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:

[email protected],

[email protected],

[email protected],

[email protected],

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.

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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

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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

immobil izat ion o f heavy metal s 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 plants Uptake 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.).

R

oo

t C

u u

pta

ke (

mg

kg

1)

S

ho

ot

Cu

up

tak

e (m

g g

-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.

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