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

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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.

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(1): 022-028.

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

  1. 1. 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 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. 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
  2. 2. 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
  3. 3. 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
  4. 4. 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). 025 Journal of Research in Agriculture (2011) 1: 022-028 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.).
  5. 5. 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, Journal of Research in Agriculture (2011) 1: 022-028 026 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 )
  6. 6. 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. 027 Journal of Research in Agriculture (2011) 1: 022-028 Ndiaye et al.,2011
  7. 7. 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. Journal of Research in Agriculture (2011) 1: 022-028 028 Ndiaye et al.,2011 Submit your articles online at www.jagri.info Advantages  Easy online submission  Complete Peer review  Affordable Charges  Quick processing  Extensive indexing  You retain your copyright submit@jagri.info www.jagri.info/Sumit.php.

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