5.MI730-Hanna Artuti Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.7.4.5ISSN 1978-3477, eISSN 2087-8575 Vol 7, No 4, November 2013, p 167-176 *Corresponding author; Phone/Fax: +62-251-8626178, Email: hanna.artuti@gmail.com Numerous soils have been polluted by mercury (Hg) as a result of anthropogenic activities, such as amalgamation of gold in gold-mining and the agricultural soils. Hg is a toxic chemical which is not degraded because it is an element (US EPA 2009). Management of the areas, which have been exposed to either intense or diffuse Hg pollution, has therefore become a major environmental concern. Strategies oriented toward the use of plants and microbes, or both in combinations within the plant rhizosphere, have been proposed in the recent years as an effective cleanup technology in removing or stabilizing heavy metals in polluted soils. Among these microbes, arbuscular mycorrhizal A river-sand culture experiment was conducted to investigate whether arbuscular mycorrhizal (AM) colonization influenced mercury (Hg) and nutrients accumulation, and whether AM fungus (AMF) Gigaspora margarita enhance host plant sengon buto (Enterolobium cyclocarpum) tolerance to Hg. Hg was applied as HgCl at different levels (375 and 750 µM) and added to the full strength of Hoagland's solution, then applied to 2 seedlings in river-sands as growth media according to treatments. The non-mycorrhizal and mycorhizal E. cyclocarpum roots took up Hg, but its translocation to the leaves was inhibited. AM inoculation decreased significantly Hg content of roots seedlings by 70.5% from non-AM inoculation seedlings. Mycorrhizae enhanced significantly Ca and Mg uptake in shoot by 1.29- and 1.27-fold higher than non-mycorrhizal seedlings, but not enhanced significantly P uptake. Based on the roots dry weight, the tolerance index of non-mycorrhizal or mycorrhizal seedlings treated with 750 µM Hg supply was > 50%. It indicated that the seedlings can tolerate up to 750 µM Hg added. Considering the possible differences in AMF response to Hg in polluted soil from the field, it is not yet clear if Gi. margarita could be applied for phytoremediation of Hg in contaminated sites. Therefore, more work needs to be done using AMF isolates to reveal the possible application in the management of Hg contaminated soils. Key words: arbuscular mycorrhizal fungus, Enterolobium cyclocarpum, Gigaspora margarita, mercury Percobaan untuk meneliti apakah kolonisasi mikoriza arbuskula (MA) mempengaruhi akumulasi merkuri (Hg) dan unsur hara, dan apakah fungi MA (FMA) Gigaspora margarita meningkatkan toleransi tanaman inang Sengon Buto (Enterolobium cyclocarpum) terhadap Hg telah dilakukan. Perlakuan Hg diberikan dengan konsentrasi berbeda (375 dan 750 μM HgCl ) yang ditambahkan ke dalam larutan hara Hoagland full strength, 2 kemudian diberikan pada semai di media tumbuh pasir sungai sesuai perlakuan. Akar E. cyclocarpum tidak bermikoriza dan bermikoriza menyerap Hg, tetapi translokasinya ke daun dihambat. Inokulasi MA nyata menurunkan kadar Hg akar semai 75% dari semai yang tidak diinokulasi MA. Mikoriza nyata meningkatkan serapana Ca dan Mg di tajuk 1.29 dan 1.27 kali lebih tinggi dari semai tidak bermikoriza, tetapi tidak nyata meningkatkan serapan P. Berdasarkan bobot kering akar, indeks toleransi semai tidak bermikoriza dan bermikoriza yang diberi perlakuan Hg 750 µM lebih dari 50%. Hal ini mengindikasikan bahwa semai dapat toleran Hg hingga 750 µM. Pertimbangan pada kemungkinan perbedaan respons FMA terhadap Hg di tanah tercemari dari lapangan, masih belum jelas apakah Gi. margarita dapat diaplikasikan untuk fitoremediasi lahan terkontaminasi Hg. Oleh karena itu, penelitian lebih lanjut dengan memanfaatkan isolat-isolat FMA diperlukan untuk mengembangkan kemungkinan aplikasinya dalam pengelolaan tanah-tanah terkontaminasi Hg. Kata kunci: Enterolobium cyclocarpum, fungi mikoriza arbuskula, Gigaspora margarita, merkuri The Role of Arbuscular Mycorrhizal Fungus (Gigaspora margarita) on Mercury and Nutrients Accumulation by Enterolobium cyclocarpum Seedlings 1,2 3 4 HANNA ARTUTI EKAMAWANTI , YADI SETIADI *, DIDY SOPANDIE , 5 AND DWI ANDREAS SANTOSA 1 Tropical Silviculture Major, Post-graduate School, Institut Pertanian Bogor, Jalan Lingkar Akademik Kampus IPB Darmaga, Bogor 16680, Indonesia; 2 Faculty of Forestry, Universitas Tanjungpura, Jalan Imam Bonjol, Pontianak, Indonesia; 3 Laboratory of Forest Biotechnology, Research Center for Biological Resources and Biotechnology, Institut Pertanian Bogor, Jalan Lingkar Akademik Kampus IPB Darmaga, Bogor 16880, Indonesia; 4 Department of Agronomy and Horticulture, Institut Pertanian Bogor, Jalan Meranti, Kampus IPB Darmaga, Bogor 16680, Indonesia; 5 Laboratory of Biotechnology Soil and Environment, Department of Soil and Land Resources, Institut Pertanian Bogor, Jalan Meranti, Kampus IPB Darmaga, Bogor 16680, Indonesia fungi (AMF) are particular interest due to their unique position at the soil/root interface and their recognized role in element transport and immobilization (Smith and Read 2008). AMF can tolerate a wide range of metal concentrations in soils (González-Guerrero et al. 2008), affect the accumulation of metals such as Cu, Cd, Zn, and As by plants and enhance the tolerance of host plants to these metals exposure in soil (González- Chávez et al. 2004; Janoušková et al. 2006; Marques et al. 2006). However, few studies have addressed the interaction between AMF and Hg in growth medium- or soil-plant system. Therefore, the potential of AMF has not yet been fully explored with respect to its Hg phytoremediation. The first report about the effects of AM inoculation on Hg behavior in soil-plant system is done by Yu et al. (2010) and it has been shown that Hg uptake was lower by mycorrhizal roots of maize than by non-mycorrhizal roots. Hg accumulation in several plants has been studied, such as white clover (Greger et al. 2005), alfalfa (Ortega-Villasante et al. 2005), lentil, chickpea (Rodríguez et al. 2007) and common vetch (Sierra et al. 2008). Nevertheless, very few studies have been conducted on the Hg tolerance in tropical trees, such as Sengon Buto (Enterolobium cyclocarpum). This species is one of the largest trees in the dry forest formation and a nitrogen-fixing species (World Agroforestry Centre 2013), therefore it could be a soil improver. If this tree species is proposed for phytoremediation on Hg-polluted soil, plant Hg uptake and resistance response should be evaluated under controlled conditions, prior to field establishment a screen to identify suitable candidate species. The interaction between AMF, tree species (such as E. cyclocarpum) and Hg was the subject of this study because of the possibility of the beneficial effect of mycorrhizae in improving the resistance of plants against Hg toxicity. The study was carried out to determine the Hg and nutrient accumulation in E. cyclocarpum seedlings inoculated with AMF Gigaspora margarita (collection of Laboratory of Forest Biotechnology, Research Center for Biological Resources and Biotechnology, Bogor Agricultural University) grown in artificial Hg-polluted river sands media in pot experiment. Such knowledge would help clarify the potential of tree species E. cyclocarpum and AMF as phytoremediation agents of Hg-polluted soils. MATERIALS AND METHODS Plant Culture in River Sands at Different Rates of Hg. E. cyclocarpum seeds were surface-sterilized by 168 EKAMAWANTI ET AL. Microbiol Indones immersing the seeds in 0.05% NaOCl for 60 sec and then rinsing them three times with distilled water (dH O). To break dormancy, the seeds were soaked in 2 boiling water for 5 min and then in dH O overnight. 2 The seeds were germinated on zeolite media. After formation of completed leaves, a seedling was transplanted on to zeolite media in polybag (15 cm x 25 cm). Mycorrhizal seedlings were obtained by inoculating the seedlings with Gi. margarita inoculums (Laboratory of Forest Biotechnology, Research Center for Biological Resources and Biotechnology, Bogor Agricultural University collection) as first inoculation First inoculation of AMF inoculum when they were germinated and transplanted on zeolite. Each nine-months old seedlings of non-mycorrhizal and mycorrhizal was grown in a PVC pot (10 cm in diameter and 30 cm in height), containing 5 kg coarse river sands (1-2 mm) as substrate and maintained in screen house. Gi. margarita inoculum (25 g) was inoculated to mycorhizal seedlings for second inoculation and sterilized mycorrhizal inoculum (25 g) and its filtrate (10 mL each pot) was applied to non-mycorrhizal seedlings. All seedlings were maintained by adding the nutrient solution for 8 weeks. The composition of the -1 nutrient solution was as follows (mmol L ) (Zornoza et al. 2010): Ca(NO ) 1.5; KNO 4.0; KH PO 1.5 and 3 2 3 2 4 -1 MgSO 1.0. Micronutrients were supplied (µmol L ): 4 Fe-EDDHA 36; MnSO .H O 33; ZnSO .7H O 1.6; 4 2 4 2 CuSO .5H O 1.6; H BO 46, and (NH ) Mo O .4H O 4 2 3 3 4 6 7 24 2 0.1. The pH of nutrient solution ranged for 5.5 to 6.0. A factorial experiment with completely randomized design with three replicates for each treatment was used. Hg treatments were 0, 375 and 750 µM, supplied as HgCl salt and applied to E. cyclocarpum seedlings, 2 either with or without AM inoculation treatments. These concentration of Hg treatments were threshold concentration of Hg toxicity for E. cyclocarpum seedlings based on the result of previous research. The experiment was carried out in screen house of Forest Microbiology Laboratory, Agency for the Research of Forest Rehabilitation and Conservation, Bogor, under the following conditions: 28 °C-33 °C temperature and 80%-95% humidity. Harvesting. Plants were harvested after 30 d to Hg exposure and divided into roots, stems and leaves. All plant material was thoroughly washed with tap water followed by a subsequent rinse in 20 mM EDTA solution for 1 min and then in deionized water for 2 min. Total fresh weight of each tissue was determined, then a representative plants were oven-dried to constant weight at 70 °C for 3 d prior to preparation for measuring dry weights and nutrients analysis, and at 50 °C prior to preparation for Hg analysis. Water content was determined based on the difference of fresh weight and dry weight. The rest of the material plants was frozen in liquid N and stored at -20 °C before 2 malondialdehyde (MDA) concentration analyzing. Analytical determination. Dried plant sample was acid digested using 25 mL of deionized water, 3.5 mL of concentrated HNO and 0.5 mL of HClO , added 3 4 to 0.5 g dry weight (DW) in a test tube. The digested solution was analyzed for Hg concentration using inductively coupled plasma-optical emission spectrometry (ICP-OES, Spectro Genesis) excitation, and calibration was performed by using standard Hg solution (Han et al. 2006).Total Hg and nutrient (P, Ca, and Mg) concentration were determined by ICP-OES. Lipid peroxidation in plant tissues was based on an estimate of malondialdehyde (MDA) concentration, as described by Heath and Packer (Ortega-Villasante et al. 2005). AM colonization was determined following Brundrett et al. (1996). Calculation. Some variables were calculated to study the resistance to mercury stress of E. cyclocarpum seedlings (Moreno-Jiménez et al. 2007): where [Hg] was Hg concentration, and DW was the roots or leaves dry weight. Nutrients (P, Ca and Mg) uptake and translocation were calculated as (Wang and Greger 2004): where DW was the entire-plant dry weight. Tolerance index (TI) of mycorrhizal and non- mycorrhizal seedlings to Hg treatments was determined based on roots dry weight, as (Rabie 2005): Water content (WC) of leaves, stems or roots was calculated as: where FW was fresh weight. Statistical analyzes. A statistical analyzes for means comparison was carried out using ANOVA and least significant difference (LSD) test using statistical software CoStat 6.400. RESULTS Mercury Accumulation. Hg accumulation in E. cyclocarpum seedlings was affected significantly by Hg treatment. Hg concentration in roots of E. cyclocarpum seedlings was 8- to 16-fold and Hg content in roots was 3- to 6-fold higher than the control (without Hg) (Table 1). However, at the 750 µM of Hg, Hg accumulation in roots was decreased significantly by 49.6% of Hg concentration and 57.2% of Hg content when compared to Hg accumulation in roots of seedlings treated with 375 µM of Hg. Hg accumulation in roots without Hg treatment shows that there was Hg cross contamination in the air. Table 1 shows that almost there are not Hg accumulation in leaves of E. cyclocarpum. On the other hand, AM inoculation decreased Hg content of roots seedlings by 70.5% from non-AM inoculation seedlings (data not shown). Nutrients Accumulation and Distribution. The highest Hg concentration had stronger effects regarding nutrients translocation than regarding nutrients uptake, especially in mycorrhizal seedlings (Table 2). The supply of 750 µM Hg caused reducing in P and Mg translocation by 53% and 47%, respectively, lower than the control (without Hg supply) in mycorrhizal seedlings. P uptake also followed a similar response to highest Hg rate by 29% lower than the control in seedlings inoculated with AMF. In contrast, nutrients (P, Ca, and Mg) uptake and translocation were not difference in non-mycorrhizal seedlings treated with the Hg supply. Moreover, nutrients uptake and translocation did not suffer decreases in seedlings at the lower Hg supply (375 µM), either in non-mycorrhizal or mycorrhizal seedlings. Table 2 also showed that AM inoculation had negative effects regarding nutrients translocation, but not regarding nutrients uptake in seedlings treated with the Hg supply. The AM inoculation caused decreases significantly in P and Mg translocation to leaves by 56% and 44%, respectively, lower than in non- mycorrhizal seedlings treated with 750 µM Hg apply. In contrast, P and Mg uptake showed no difference effects of between control (no AM inoculation) and AM inoculation in seedlings at 375 µM Hg supply. This Volume 7, 2013 Microbiol Indones 169 -1 Hg content in roots (ng roots ) -1 and leaves (ng leaves ) = [Hg] × g DW -1 Nutrient uptake (nmol g DW) -1 nmol plant g DW = -1 Nutrient translocation (nmol Hg g DW) -1 nmol leaves g DW = TI (%) DW of roots at Hg treated DW roots at non Hg treated of the same treatments = × 100 WC (%) (FW-DW) of leaves, stems, or roots FW of leaves, stems, or roots = × 100 supply (Table 4). Dry weight of leaves, stems and roots decreased in the range 38-54%, 54-67%, and 43-54%, respectively. It indicated that there was reduction of plant growth caused by Hg supply up to 750 µM during 30 d of Hg exposure. On the other hand, biomass of seedling treated with AM inoculation was decreased significantly (Table 5). In stems and roots, E. cyclocarpum seedlings treated with AM inoculation showed 63% and 68% decrease in the biomass respectively, but a sharp decrease (72%) was occurred in leaves. Regarding water content (Table 6), the effect of AM inoculation depended on Hg treatment. At control (without Hg supply), leaves and stems water content in tendency was also found regarding Ca uptake and translocation. On the other hand, AM inoculation increased P uptake, significantly, and tended to increase Ca and Mg uptake and also nutrients translocation in seedlings treated without Hg supply. In addition, AM inoculation as a single factor treatment also enhanced Ca and Mg uptake in seedlings, significantly, by 1.29- and 1.27-fold higher than non- mycorrhizal seedlings, respectively (Table 3). E. cyclocarpum Seedlings Biomass and MDA Concentration. The negative effect of Hg in plants was reflected in biomass (dry weight), being significantly (p < 0.05) reduced in non-mycorrhizal and mycorrhizal seedlings, when cultivated with Hg Microbiol Indones170 EKAMAWANTI ET AL. Hg (µM) -1 Hg concentration (ng g DW) Hg content (ng) Roots Leaves Roots Leaves 0 38.2 ± 26.2 a 1.8 ± 3.0 363.5 ± 467.4 a 9.6 ± 14.7 375 611.0 ± 124.3 c n.d 2,066.5 ± 1,759.0 c n.d 750 302.8 ±154.8 b n.d 1,183.0 ± 991.1 b n.d Hg (µM) AM inoculation Without With With out With -1------ P uptake (µmol g DW) ------- ------- P translocation (µmol g-1 DW) -------- 0 109.8 ± 23.3 a(B) 164.7 ± 14.8 a(A) 19.3 ± 7.3 a(A) 29.2 ± 8.3 a(A) 375 127.0 ± 8.1 a(A) 162.5 ± 21.5 a(A) 23.4 ± 6.0 a(A) 28.5 ± 16.1 a(A) 750 142.1 ±36.7 a(A) 116.2 ±21.2 b(A) 30.2 ± 0.7 a(A) 13.3 ± 2.5 b(B) -1-------- Ca uptake (µmol g DW) -------- -1 -------- Ca translocation (µmol g DW) ------- 0 60.0 ± 3.3 a(A) 84.3 ± 14.3 a(A) 65.1 ± 21.8 a(A) 93.4 ± 30.5 a(A) 375 47.7 ± 2.6 a(A) 60.9 ± 12.2 a(A) 54.1 ± 10.3 a(A) 61.7 ± 18.9 a(A) 750 50.0 ± 3.2 a(A) 58.7 ± 3.6 a(A) 68.6 ±15.0 a(A) 42.6 ± 11.6 a(A) -1-------- Mg uptake (µmol g DW) -------- -1 ------- Mg translocation (µmol g DW) ------- 0 23.3 ± 2.3 a(A) 34.5 ± 8.6 a(A) 67.9 ± 19.7 a(A) 102.4 ± 32.1 a(A) 375 24.4 ± 1.3 a(A) 31.5 ± 2.2 a(A) 75.5 ± 15.5 a(A) 88.6 ± 31.1 a(A) 750 23.3 ±6.4 a(A) 24.6 ± 7.3 a(A) 83.6 ± 3.8 a(A) 46.9 ± 8.3 b(B) Table 1 Hg concentration and Hg content in roots and leaves of E. cyclocarpum seedlings grown for 30 d in river sands with treatment of different Hg concentration Table 2 Nutrients uptake and translocation in E. cyclocarpum seedlings grown for 30 d in river sands with treatments of different Hg concentrations and AM inoculation Significant differences among Hg treatments are indicated by different letter (mean ± SD, n = 6; LSD’s test, p < 0.05). DW: dry weight. Significant differences among Hg treatments are indicated by small different letter and among AM inoculation treatments with different capital letters (mean ± SD, n = 3; LSD’s test, p < 0.05). DW: dry weight. Volume 7, 2013 Microbiol Indones 171 mycorrhizal seedlings were increased by 11% each. Even though AM inoculation did enhance leaves water content, it increased stems water content significantly by 27% when the seedlings were exposed to 750 µM Hg. Moreover, mycorrhizal inoculation enhanced roots water content, 17% higher than non-mycorrhizal, in seedlings treated with or without Hg treatments (data not shown). Hg exposure for 30 d increased MDA concentration in roots of E. cyclocarpum seedlings, significantly (Fig 1). MDA concentration of roots increased when the seedlings were grown in Hg-treated river-sand media, either with or without AM inoculation. AM inoculation caused the highest increase in MDA concentration by 485% in the roots of seedlings treated with 750 µM Hg, when compared to the control (without AM inoculation). On the other hand, 375 µM and 750 µM Hg supply resulted in the increased MDA concentration in leaves by 14% and 97%, respectively (data not shown). Tolerance index of E. cyclocarpum seedlings grown for 30 d in river sands treated with 375 µM Hg was < 50% but the tolerance index was > 54% when the seedlings were treated with 750 µM (Table 7). Roots of non-inoculated seedlings remained non- mycorrhizal while roots of inoculated ones were colonized by AMF (Table 7). The low AMF colonization in seedlings treated with Hg indicated that Gi. margarita colonization was reduced in the presence of Hg in substrate. DISCUSSION The results obtained during this research have shown that AMF Gi. margarita decreased Hg content in root seedlings. We predicted that decreasing Hg content in roots is one of Hg tolerance mechanisms in mycorrhizal symbiosis. This result was in line with Yu et al. (2010), AMF Glomus mosseae decreased significantly Hg concentration in maize roots when Hg -1 was applied at the rates of 2.0 dan 4.0 mg kg . All interactions of Hg with soil/growth media, roots, and AMF influence its uptake by plants, but this presume need to be investigated in further research. On the other hand, Hg accumulation in roots treated with 750 µM of Hg supply decreased significantly by 49.6% of Hg concentration and 57.2% of Hg content when compared to Hg accumulation in roots of seedlings treated with 375 µM of Hg. It shows that at the highest Hg treatment, Hg accumulation in root seedlings was limited. This result was in contrast with Esteban et al. (2008) result, which found the pattern of long-term (over 28 d) Hg accumulation in shoots and roots white lupin (Lupinus albus L.) can be dissected into linear and hyperbolic (saturable) components, when they were grown in 5 and 10 µM Hg supply in hydrophonics. The result of experiment also showed that a main part of Hg accumulated by E. cyclocarpum AM inoculation P uptake -1 (µmol g DW) Ca uptake -1 (µmol g DW) Mg uptake -1 (µmol g DW) Without 126.3 ± 26.2 a 52.6 ± 6.3 b 23.7 ± 3.5 b With 147.8 ± 29.1 a 68.0 ± 15.6 a 30.2 ± 7.2 a Hg (µM) Dry weight (g) Roots Stems Leaves 0 7.0 ± 4.8 a 11.1 ± 5.6 a 3.7 ± 2.2 a 375 3.2 ± 2.5 b 3.7 ± 2.7 b 1.7 ± 1.6 b 750 4.0 ± 2.9 b 5.1 ± 4.4 b 2.3 ± 2.1 b Table 3 Nutrients uptake in E. cyclocarpum seedlings grown for 30 d in river sands with treatment of mycorrhizal Table 4 Leaves, stems and roots dry weight of E. cyclocarpum seedlings grown for 30 d in river sands with treatments of different Hg concentrations Significant differences among Hg treatments are indicated by different letter (mean ± SD, n = 9; LSD’s test, p < 0.05). DW: dry weight. Significant differences among Hg treatments are indicated by different letter (mean ± SD, n = 6; LSD’s test, p < 0.05). DW: dry weight. growth and nutrient uptake (Smith et al. 2008). However, AM symbiosis decreased nutrients (P and Mg) translocation in seedlings treated with the highest Hg supply (750 µM). We predict that disturbances of water fluxes altered P and Mg distribution due to high concentration Hg in growth substrate and also due to the existence of P and Mg competition between plant and AMF Gi. margarita in a limited space of pot experiment. Although a contribution of concentration effects after Hg supply cannot be ruled out, the increase of some nutrients could also be a strategy to avoid toxicity in plants (Moreno-Jiménez et al. 2007). In other case, the increase of nitrogen by nitrogen supply prevents oxidative stress in roots (Carrasco-Gil et al. 2012). Hg can induce toxicity symptoms in plants such as inhibition of plant growth or disturbances on water and nutrient uptake (Patra at al. 2000). Mercury accumulated in plants evokes severe phytotoxicity and impairs numerous metabolic processes including nutrient uptake, water status, and photosynthesis (Chen et al. 2012). The inhibition of plant growth might be the first symptom of Hg stress (Cho et al. 2000). It was showed that Hg decreased significantly dry weight of seedlings was in the roots. There was no translocation of Hg to the aerial part (leaves) even though the Hg concentration in the growth media was higher. It indicates that E. cyclocarpum is not an Hg accumulator, but an Hg excluder. This result in line with some studies which have indicated that only a very small amount of Hg is translocated to plant shoot after root uptake and Hg in leaf mainly comes from the uptake of air Hg (Ericksen et al. 2004; Greger et al. 2005; Fay et al. 2007; Chen et al. 2009). Even though it was not an Hg accumulator, the potential of E. cyclocarpumas an Hg phytoremediation agent could be explored because the roots accumulated Hg. Plants that accumulate Hg in the roots convert the pollutant into less available forms, thus they could be used to prevent Hg leaching and moving to other place. In contrast, arbuscular mycorrhizal symbiosis tend to enhance nutrients (P, Ca, and Mg) uptake and translocation, especially in seedlings treated without Hg supply and 375 µM Hg supply (Table 2). Nevertheless the enhancement was not significant but as a single factor, AM inoculation enhanced P, Ca and Mg uptake, significantly (Table 3). It is well documented that AM symbiosis can increase plant Microbiol Indones172 EKAMAWANTI ET AL. AM inoculation Dry weight (g) Roots Stems Leaves Without 7.2 ± 3.7 a 9.6 ± 5.1 a 3. 9 ± 1.9 a With 2.3 ± 1.4 b 3.6 ± 3.6b 1.1 ± 0.9 b Hg (µM) AM inoculation Without With Without With Without With Water content (%) --------------Roots -------------- -------------- Stems --------------- ------------- Leaves -------------- 0 56.1 ± 9.3 a(A) 73.3 ± 3.3 a(A) 61.3 ± 2.8 b(B) 68.0 a ± 2.1 a(A) 68.3 ± 2.9 b(B) 75.8 ± 0.3 a(A) 375 66.3 ± 4.0 a(A) 74.9 ± 7.2 a(A) 67.1 ± 2.8 a(A) 66.7 a ± 2.6 a(A) 73.3 ± 1.2 a(A) 72.3 ± 4.0 a(A) 750 69.2 ± 9.8 a(A) 76.3 ± 3.3 a(A) 61.1 ± 1.7 b(B) 77.6 a ± 9.9 a(A) 69.5 ± 0.5 ab(A) 68.5 ± 2.3 b(A) Table 5 Leaves, stems and roots dry weight of E. cyclocarpum seedlings grown for 30 d in river sands with treatments of AM inoculation Table 6 Roots, stems, and leaves water content of E. cyclocarpum seedlings grown for 30 d in river sands with treatments of different Hg concentrations and AM inoculation Significant differences among Hg treatments are indicated by different letter (mean ± SD, n = 9; LSD’s test, p < 0.05) Significant differences among Hg treatments are indicated by small different letter and among AM inoculation treatments with different capital letters (mean ± SD, n = 3; LSD’s test, p < 0.05) Volume 7, 2013 Microbiol Indones 173 of roots, stems and leaves significantly (Table 5), even though AM inoculation increased P, Ca, and Mg uptake (Table 3). We predicted that increase in P, Ca, and Mg uptake might be not followed by increase in other macro and micro nutrients. Therefore, increase in P, Ca and Mg uptake was not followed by increase in plant dry weight. Additionally, growth depressions can be explained with respect to carbon (C) demand and P supply by the AMF, where C drain to AMF was not roots, stems, and leaves of E. cyclocarpum seedlings during 30 d exposure to Hg supply (Table 4). The reduction of plant growth caused by Hg also been shown for Rumex induratus and Marrubium vulgare (Moreno-Jiménez et al. 2007). Generally, the formed mycorrhizal symbiosis significantly improved plant growth performance, such as plant height, stem diameter, shoot, root or total dry weight. In contrast, as a single factor of AM inoculation decreased dry weight a (A) b (A) b (A) a (A) b (A) c (B) 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0 375 750 M D A c o n ce n tr at io n ( n m o l g -1 r o o t F W ) Hg treatment (µM) Without AM With AM Fig 1 MDA concentration in roots of E. cyclocarpum seedlings with different Hg concentration (mean ± SD, n = 3). Significant differences among Hg treatments are indicated by small different letter and among AM inoculation treatments with different capital letters (LSD’s test, p<0.05). Table 7 Tolerance index and AM colonization of E. cyclocarpum seedlings grown for 30 d in river sands with treatments of different Hg concentrations and AM inoculation Hg (µM) AM inoculation Without With Without With ------------ Tolerance index* (%) ------------ ------ AM colonization (%) ----- 0 100 100 0 65.9 375 46.3 45.6 0 47.2 750 54.2 66.9 0 40.8 Remark: * The tolerance index was based on roots dry weight imbalance to P supply from AMF (Grace et al. 2009). Kiers et al. (2011) stated that plants can detect, discriminate, and reward the best fungal symbionts with more carbohydrates and in turn, their fungal symbionts enforce cooperation by increasing nutrient transfer only to those roots providing more carbohydrates. It has been addressed that Hg can disturbed water fluxes, therefore, it can alter nutrients distribution which depends on water movement within the xylem (Moreno-Jiménez et al. 2007). Considering now water content, in each organ of E. cyclocarpum seedlings did not suffer reduction in water content during Hg exposure. It indicated that water movement within the seedlings was not disturbed by Hg apply. Therefore, nutrients distribution (P, Ca, and Mg) in E. cyclocarpum seedlings were not inhibited, especially when the seedlings exposed to Hg up to 375 µM concentration. This research only analyzed P, Ca, and Mg concentration, not other nutrients. Therefore, we cannot state if other nutrients translocation is affected. The role of AM inoculation to increase the water content of roots might be as an indirect strategy of seedlings to overcome Hg-induced water stress when the roots contacted Hg directly. It has been known, extraradical hyphae of AMF can also increase water uptake in addition to nutrients uptake (Smith et al. 2008). MDA is one of organic compounds has been used as a biomarker, which can be useful in the early diagnosis of metal toxicity (Prasad 2003). In our research, AM inoculation enhanced MDA concentration in roots, significantly, higher than non-mycorrhizal seedlings when exposed to the highest Hg (750 µM) treatment. It suggested some degree of oxidative stress, probably due to the high Hg concentration in the roots without any increase of the antioxidant (Moreno-Jiménez et al. 2007) in roots of mycorrhizal seedlings. In contrast, MDA concentration in roots of mycorrhizal seedlings was not different with roots of non-mycorrhizal seedlings when exposed to the lower Hg (375 µM) supply. Increases of MDA concentration by Hg exposure have been observed in some previous results research (Cho et al. 2000; Ortega-Villasante et al. 2005; Moreno-Jiménez et al. 2006; Esteban et al. 2008). However, it has not clear yet how its mechanism, especially in mycorrhizal plants when they were exposed to Hg and there is no information available, therefore further investigation is still needed. In other case, inoculation with AMF caused reduction in MDA content in comparison to salinized plants, indicating lower oxidative damage in the colonized plants (Latef et al. 2011). In our research, there was no effect of AM inoculation on MDA concentration in leaves but only Hg supply as a single factor resulted in the increased of MDA concentration in leaves, nevertheless, there was no Hg accumulation in leaves. It was probably due to indirect effect of Hg accumulation in roots. This presumption has to be proved in further study. Roots of inoculated plants were colonized by AMF, while non-inoculated controls remained non- mycorrhizal (Table 7). However, the percentage of AM colonization was reduced in the presence of Hg. This result indicated that the concentration of Hg supply in the substrate was harmful to AMF Gi. margarita. This finding is in line with Rabie (2005) who reported that sensitivity of AM symbionts to heavy metal polluted soil expressed as a reduction in root colonization. In contrast, addition of Hg in soil did not significantly influence maize root colonization rate (Yu et al. 2010). Another interesting result showed at Table 7, the presence of AMF could increase the metal tolerance index of E. cyclocarpum seedlings compared with non- mycorrhizal seedlings when they treated with 750 µM Hg. This result emphasizes that AMF could be potentially effective in protecting seedlings exposed to high levels of Hg concentration. The AMF ability to alleviate heavy metals stress of plants grown in heavy metal polluted soil was previously proved by Rufyikiri et al. (2000); Hildebrandt et al. (2007). In contrast, tolerance index of mycorrhizal or non-mycorrhizal seedlings grown for 30 d in river sands treated with 375 µM Hg was lower than seedlings treated with 750 µM (Table 7). It might be related to Hg accumulation in roots treated with 375 µM Hg was higher than in roots treated with 750 µM Hg (Table 1) then resulted in the decreased of roots dry weight. In conclusion, this study provides further evidence for the protective effects of AMF Gi. margarita on E. cyclocarpum against Hg contamination, i.e. non significant effect on Hg accumulation in roots, a tendency to increase nutrient uptake and translocation when seedlings treated with Hg supply up to 375µM, the sharp decrease of dry biomass in seedlings, no different effect on water content in seedlings treated with Hg supply and no increase in MDA concentration in seedlings treated with 375 µM. It suggested that the role of symbiosis AMF Gi. margarita and E. cyclocarpum seedlings can tolerate up to 375 µM Hg supply. Even though, it is not yet clear if Gi. margarita could be applied for phytostabilization of Hg in contaminated sites. 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