A nonpathogenic Fusarium oxysporum strain enhances phytoextraction of heavy metals by the hyperaccumulator Sedum alfredii Hance

Journal of Hazardous Materials 229–230 (2012) 361–370

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A nonpathogenic Fusarium oxysporum strain enhances phytoextraction of heavy metals by the hyperaccumulator Sedum alfredii Hance Xincheng Zhang a , Li Lin c , Mingyue Chen b , Zhiqiang Zhu a,d , Weidong Yang a , Bao Chen a , Xiaoe Yang a,∗ , Qianli An b,∗∗ a MOE Key Laboratory of Environment Remediation and Ecosystem Health, College of Environmental and Resources Science, Zhejiang University, Zijingang Campus, Hangzhou 310058, China b Institute of Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou 310058, China c Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, China d Agricultural College, Hainan University, Renmin Road 58, Haikou 570228, China

h i g h l i g h t s I I I I

A culturable Fusarium fungus enhanced metal extraction by host hyperaccumulator. A new way of fungal-assisted phytoextraction by nonmycorrhized hyperaccumulators. A new way using indigenous fungi other than AMF to enhance phytoextraction. A new function and application potential for nonpathogenic Fusarium fungi.

a r t i c l e

i n f o

Article history: Received 8 March 2012 Received in revised form 7 June 2012 Accepted 8 June 2012 Available online 16 June 2012 Keywords: Heavy metal Phytoremediation Hyperaccumulator Sedum alfredii Hance Fusarium oxysporum

a b s t r a c t Low biomass and shallow root systems limit the application of heavy metal phytoextraction by hyperaccumulators. Plant growth-promoting microbes may enhance hyperaccumulators’phytoextraction. A heavy metal-resistant fungus belonged to the Fusarium oxysporum complex was isolated from the Zn/Cd co-hyperaccumulator Sedum alfredii Hance grown in a Pb/Zn mined area. This Fusarium fungus was not pathogenic to plants but promoted host growth. Hydroponic experiments showed that 500 ␮M Zn2+ or 50 ␮M Cd2+ combined with the fungus increased root length, branches, and surface areas, enhanced nutrient uptake and chlorophyll synthesis, leading to more vigorous hyperaccumulators with greater root systems. Soil experiments showed that the fungus increased root and shoot biomass and S. alfredii-mediated heavy metal availabilities, uptake, translocation or concentrations, and thus increased phytoextraction of Zn (144% and 44%), Cd (139% and 55%), Pb (84% and 85%) and Cu (63% and 77%) from the original Pb/Zn mined soil and a multi-metal contaminated paddy soil. Together, the nonpathogenic Fusarium fungus was able to increase S. alfredii root systems and function, metal availability and accumulation, plant biomass, and thus phytoextraction efficiency. This study showed a great application potential for culturable indigenous fungi other than symbiotic mycorrhizas to enhance the phytoextraction by hyperaccumulators. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Phytoextraction is a low-cost, sustainable, and challenging in situ technique for remediation of hazardous heavy metals from contaminated soils. Efficient phytoextraction requires high levels of heavy metal availability, root uptake, root-to-shoot translocation, shoot detoxification/sequestration and high shoot biomass [1].

∗ Corresponding author. Tel.: +86 571 88982507; fax: +86 571 88982507. ∗∗ Corresponding author. Tel.: +86 571 88982255; fax: +86 571 88982255. E-mail addresses: [email protected], [email protected] (X. Yang), [email protected] (Q. An). 0304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2012.06.013

Metal hyperaccumulating plants naturally accumulate extraordinarily high concentrations of trace elements in their shoot tissues and thus appear to have ideal traits for phytoextraction. However, most of hyperaccumulators grow slowly and produce shallow root systems and low shoot biomass, limiting their application for heavy metal phytoextraction [1,2]. Therefore, new techniques that significantly increase metal bioavailability and accumulation and plant biomass would enhance the efficacy of phytoextraction by hyperaccumulators for future phytoremediation application. Numerous recent studies have clearly shown that plant growthpromoting bacteria and fungi mainly arbuscular mycorrhizal fungi (AMF) can promote plant growth and increase heavy metal

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bioavailability and thus facilitate phytoextration by hyperaccumulators or non-hyperaccumulators [3–6]. Plant growth-promoting bacteria promote plant growth by modulating plant hormone levels and enhancing nutrient uptake [4–7]. Bacterial production of organic acids and siderophores increases phosphate and iron nutrient availabilities and also heavy metal availabilities [5,7]. AMF can promote host growth by enhancing water and nutrient uptake. Besides extensive extraradical hyphal networks, certain mycorrhizal plants establish modified root architecture and topology, generally a greater root system with longer and more branched roots, facilitating uptake of water, nutrients, and metal elements [8]. Typically, microorganisms that enhance metal phytoextraction have been isolated and selected according to their ability to tolerate some metals [3]; metal-resistant bacteria and AMF promote plant growth and phytoextraction when lower and less toxic levels of heavy metals are present [5,9]. Sedum alfredii Hance is a vegetatively propagated perennialof the Crassulaceae family and was first found in an old Pb/Zn mined area in Quzhou, Zhejiang Province, southeast of China [10,11]. S. alfredii is a relatively fast-growing, high-biomass Zn/Cd cohyperaccumulator and Pb accumulator and can accumulate up to 2% Zn (w/w) and 0.9% Cd (w/w) in shoots and 1.4% Pb (w/w) in roots grown hydroponically without showing toxicity symptoms. Its growth is slightly promoted by Zn2+ , Cd2+ , or Pb2+ at the levels of 125–500 ␮M, 25–100 ␮M, or 60–480 ␮M, respectively [10–12], which are toxic to the non-hyperaccumulating ecotype S. alfredii plants [12–14]. S. alfredii has now been used as a model plant to study metal hyperaccumulation and develop new techniques to enhance phytoextraction [13–19]. Indigenous root-associated metal-resistant bacteria have been shown to significantly increase S. alfredii growth and heavy metal uptake and root-to-shoot translocation [19,20]. Wu et al. found low to moderate levels (8.5–45.8%) of colonization of S. alfredii by AMF at uncontaminated and metalcontaminated sites and no significant effects of AMF colonization on heavy metal accumulation [21]. We aim to use indigenous plant growth-promoting microbes to enhance S. alfredii phytoextraction of heavy metals. Here, we isolated a metal-resistant fungus belonged to the Fusarium oxysporum complex from the S. alfredii plants grown in the Pb/Zn mined area. Firstly, we investigated the fungal effects on hydroponic S. alfredii growth and Zn/Cd hyperaccumulation and fungal pathogenicity to plants. Secondly, we investigated the application potential of the culturable non-mycorrhizal fungus to enhance S. alfredii phytoextraction from multi-metal contaminated soils. 2. Materials and methods 2.1. Plants The hyperaccumulating ecotype S. alfredii plants grown at the vegetable stage were collected from three sites in an old Pb/Zn mined area in Quzhou, Zhejiang Province, China. Heavy metal concentrations in the S. alfredii plants used for cutting propagation and microbial isolation are presented in Table 1. 2.2. Microbial isolation Whole plants were surface sterilized with 70% ethanol for 3 min twice and 2% active chloride for 3 min and washed with sterile water for six times. Plant stems were cut transversely into pieces of approximately 1 cm in length and put into 1.5 mL sterile eppendorf tubes. Intercellular fluid was obtained by centrifugation at 3 000 g and serially diluted with sterile water; 100 ␮L of each diluted solution was spread onto modified DF agar media [22] containing 1/10 strength of phosphate, 3 mM 1-aminocyclopropane-1-carboxylate

as the sole N source, and 20 mM ZnSO4 , 2 mM Cd (NO3 )2 or 10 mM Pb (NO3 )2 , respectively, or the mixtures of the above heavy metals. After incubation at 28 ◦ C for 7 d, individual bacterial and fungal isolates were purified with the isolation media for three times. Purified fungal isolates were transferred on the potato dextrose agar (PDA). Minimal inhibitory concentrations of heavy metals to fungal isolates were determined with the PDA containing increasing concentrations of metals; fungal cultures were incubated at 28 ◦ C for 7 d and compared with cultures on PDA without supplemented metals. 2.3. Fungal identification Fungal colonies were observed with eyes. Fungal spores were observed with a Leica CTR 5000 microscope (Leica Microsystems, Wetzlar, Germany). Genomic DNA of 3-d old fungal mycelia was extracted with the method described by Timberlake and Marshall [23]. PCR amplifications of the internal transcribed spacer region (ITS) and translation elongation factor (TEF) were performed with the primers ITS1/ITS4 [24] and ef1/ef2 [25]. The respective amplification products of 550 bp and 650 bp were purified, cloned and sequenced as previously described [26]. The obtained putative ITS and TEF sequences of the fungal isolate SaCS12 were BLASTed [27] and deposited in the GenBank database under JQ039192 and JQ039193, respectively. 2.4. Phytopathogenic test Mycelia of the SaCS12 fungus on PDA were transferred into 200 mL liquid potato dextrose media in 500 mL-flasks and grown at 150 rpm at 25 ◦ C for 5 days. The fungal cultures were centrifuged and washed with sterile distilled water. Fungal mycelia were fragmented using a homogenizer for 15 s and diluted to a suspension with an optical density of 2.0 at 600 nm [28]. Vermiculite and perlite were mixed at 3:1 ratio by volume as substrate. One kilogram of the substrate was mixed with 10 mL of the fungal suspension for plant growth. Surface-sterilized seeds of cucumber, watermelon, tomato, pepper, soybean, pea, tobacco, cotton, and wheat were germinated and grown on wetted filter papers in Petri dishes (15 cm in diameter) at 25 ◦ C in the dark for 5–10 d. Roots of healthy seedlings were wounded by cutting off root tips and then immersed in the fungal suspensions for 1 h. Seedlings were transplanted to the substrate mixed with the fungi. Seedlings with wounding roots immersed in sterile water were used as controls and transplanted to substrates without fungal inoculation. All seedlings were maintained under 25 ◦ C, 12 h photoperiod and 80% relative humidity in growth chambers for 4 weeks. Fusarium wilt symptoms were assessed by visual inspection of the plants twice a week. 2.5. Plant preculture Approximately 5 cm shoots were cut from healthy S. alfredii plants and propagated in perlite under a 12 h light/12 h dark, 25/20 ◦ C, 70/80% relative humidity period in growth chambers for 3 months. Approximately 5 cm of new shoots were used for hydroponics and grown with a 1/10 strength nutrient solution [containing (in mM) Ca (NO3 )2 ·4H2 O, 2.00; KH2 PO4 , 0.10; MgSO4 ·7H2 O, 0.50; KCl, 0.10; K2 SO4 , 0.70; and (in ␮M) H3 BO3 , 10.0; MnSO4 ·H2 O, 0.50; ZnSO4 ·7H2 O, 1.0; CuSO4 ·5H2 O, 0.20; (NH4 )6 MoO24 , 0.01; and Fe-EDTA, 100] for 15 d. Rooted seedlings were transferred to 2.5 L half-strength nutrient solution in plastic pots and precultured for 7 d. Roots of the precultured seedlings were surface-sterilized by immersion in 70% ethanol for 30 s and then in 2% active chloride for 2.5 min and six washes with sterile water before transplanted to new solutions and soils.

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Table 1 Heavy metal concentrations in S. alfredii plants used for isolation of microbes. Sample plants

Metal concentration (mg kg−1 dry weight) Shoots

1 2 3

Roots

Zn

Cd

Pb

Zn

Cd

Pb

4096 ± 200 4511 ± 179 5252 ± 289

220 ± 12 299 ± 17 255 ± 14

176 ± 10 164 ± 11 200 ± 12

1119 ± 76 1221 ± 86 1009 ± 58

44 ± 2.9 50 ± 3.3 42 ± 2.8

1577 ± 99 1611 ± 102 1705 ± 119

2.6. Hydroponic culture, heavy metal treatments and fungal inoculation Seven seedlings were planted in 2.5 L half-strength nutrient solution in a plastic pot. Six pots of seedlings were prepared for each treatment. Seedlings were subjected to the following treatments. (i) Control: seedlings were grown in the solution added with 2.5 mL autoclaved fungal suspension prepared as described in 2.4. (ii) Fungal inoculation: seedlings were grown in the solution added with 2.5 mL fungal suspension. (iii) Zn- or Cd-treatment: plants grown in the solution containing 500 ␮MZnSO4 or 50 ␮MCd (NO3 )2 , which is toxic to non-hyperaccumulator but stimulative to hyperaccumulator [10,11,13,14], and added with 2.5 mL autoclaved fungal suspension. (iv) Zn- or Cd-treatment and fungal inoculation: plants grown in the solution containing 500 ␮M ZnSO4 or 50 ␮M Cd (NO3 )2 and added with 2.5 mL fungal suspension. Zn-related and Cd-related treatments were respectively performed with independent controls. Twenty four pots of seedlings were prepared for Zn-related or Cd-related treatments. The solutions were adjusted daily with 0.1 M HCl or NaOH to pH 5.5–5.8, aerated every day, and renewed every 3 d. 2.7. Plants grown in multi-metal contaminated soils and fungal inoculation Two types of aged multi-metal contaminated soils were used. The mined soil was obtained from the old Pb/Zn mined area where the S. alfredii plants used in this study were collected. The paddy soil was obtained from fields near an abandoned Cu smelter in Fuyang, Zhejiang province, China. Both types of soils were collected from the surface layer (0–20 cm). After air-drying for 15 d, the soils were sieved through a 2-mm sieve, mixed thoroughly and air-dried for two months. The aged soils were mixed thoroughly with the fungal suspension prepared as described in the section 2.4 or autoclaved fungal suspension at a ratio of 10 mL/kg soil. One kilogram of the soil mixtures were filled in a plastic pot. Nine seedlings prepared as described in the section 2.5 were transplanted in each pot. Six pots of seedlings were prepared for each treatment. Seedlings grown in soil mixtures with the autoclaved fungi were used as control treatments. Soil mixtures without plantation were also used for measurement of fungal effects on soil metal availability. Soil water-holding capacity of 60% (w/w) was maintained by adding deionized water every 4 days. Plants were grown in a glass house under natural light and period of 30/20 ◦ C, 70/85% relative humidity for 5 months (from March to August). After shoot harvest, roots and soils in the pots were separated using a 2–mm sieve. The sieved soils were mixed thoroughly and used for metal analyses. 2.8. Root analysis Roots of harvested plants were rinsed with tap water and then analyzed with a root automatism scan apparatus (MIN MAC STDI600+ , Tokyo, Japan) equipped with the WinRHIZO software (Regent Instruments Inc., Quebec, Canada).

2.9. Biomass determination Roots of harvested plants were washed as previously described [11] to remove excess heavy metals adhering to the root surface. Shoots and roots were separated and lyophilized with a lyophilizer (Labconco Corp., Kansas City, MO, USA) to constant weight. 2.10. Chlorophyll determination Fresh leaves of harvested hydroponic plants were frozen in liquid nitrogen, ground to powder and weighed. Chlorophylls in 0.3 g of the leaf powder were extracted with 30 mL 80% acetone. Chlorophylls were spectrophotometrically measured at 646.6 and 663.6 nm and calculated as described by Porra [29]. 2.11. Soil and elements analysis Soil texture, pH, organic matter, cation exchange capacity, total N, P, K, and available N, P, K were determined using standard methods [30]. The soil texture was determined using the hydrometer method. The soil pH was measured using the glass-electrode method in a ratio of soil to water at 1:2.5 (w/v). The soil organic matter was determined using the wet oxidation method. Cation exchange capacity was determined by repeated saturation using 1 M NH4 OAc (pH 7.0) followed by washing, distillation and titration. The soil total N, P, and K was determined using the Kjeldahl, ascorbic acid-blue color and flame photometer method, respectively. The soil available N, P and K was extracted with 2 M KCl, 0.5 M NaHCO3 , and 1 M NH4 OAc (pH 7.0), respectively, and was determined using the steam-distillation, ascorbic acid-blue color and flame photometer method, respectively. Soil samples were lyophilized, ground, weighed, and then digested by wet ashing in HNO3 –HClO4 –HF (5:1:1 by volume) mixtures to determine the total metal element concentrations. The available metal elements in soils were extracted with the Mehlich-3 soil test extractant [31]; the soil to extractant ratio was 1:10 (w/v). Shoot and root samples were lyophilized, ground, weighed, and then digested by wet ashing in HNO3 –HClO4 (5:1 by volume) mixtures. Metal concentrations were determined using an Agilent 7500a inductively coupled-plasma mass spectrometer (Agilent technologies, Santa Clara, CA, USA). Shoot and root N concentrations were determined using the Kjeldahl method after digestion of shoots and roots with H2 SO4 –H2 O2 [30]. 2.12. Calculation of translocation factor, bioaccumulation factor and phytoextraction rate The metal translocation factor (TF) was calculated by the metal concentration ratio of shoots to roots. The metal bioaccumulation factor (BAF) for Zn or Cd was defined as the metal concentration ratio of shoots to soils because shoots contained high concentrations of Zn and Cd; BAF for Pb or Cu was defined as the metal concentration ratio of roots to soils because roots contained high concentrations of Pb and Cu. The metal phytoextraction rate (PR)

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was defined as the metal mass ratio (percentage) of plants (shoots and roots) to soils before plantation.

Table 2 Root parameters of hydroponic S. alfredii seedlings treated with 500 ␮M Zn or 50 ␮M Cd, and/or F. oxysporum inoculation. Number of root tips plant−1

2.13. Statistical analysis

Treatments

Total root length (cm plant−1 )

Statistical analysis was performed using the SPSS statistical software (version 16.0). Data were tested at significant level of p < 0.05 using one-way ANOVA. Graphical work was performed using Sigma Plot (version 10.0).

Control Inoculation Zn Zn + Inoculation

720 813 893 1213

± ± ± ±

151b 75b 217b 200a

1373 1848 1541 2554

270c 149b 110bc 266a

173 237 214 319

± ± ± ±

32c 21b 40bc 60a

Control Inoculation Cd Cd + Inoculation

730 837 905 1151

± ± ± ±

100c 73bc 71b 171a

1313 ± 60c 1870 ± 171ab 1741 ± 307bc 2389 ± 699a

187 256 194 265

± ± ± ±

24b 27a 32b 37a

3. Results 3.1. Fungal isolation and characterization Several filamentous fungi were isolated from the surfacesterilized hyperaccumulating ecotype S. alfredii plants grown in the Pb/Zn mined soil. One of the fungal isolate SaCS12 could tolerate multiple heavy metals including 20 mM Zn, 10 mM Pb, 2 mM Cd and 0.8 mM Cu. SaCS12 showed the typical colony and spore morphologies of F. oxysporum (Fig. S1). Moreover, amplification, sequencing, and BLASTing of its ITS and TEF sequences revealed that SaCS12 was closely related to those fungi affiliated to the F. oxysporum complex. Therefore, SaCS12 was preliminarily identified as F. oxysporum. Pathogenicity tests showed that SaCS12 was not pathogenic to seedlings of cucumber, watermelon, tomato, pepper, soybean, pea, tobacco, cotton, and wheat (data not shown), which are often suffered from wilt and root-rot diseases caused by F. oxysporum pathogens. Moreover, SaCS12 was not pathogenic but beneficial to the host S. alfredii plants (see below). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2012.06.013.

± ± ± ±

Surface area (cm2 plant−1 )

Values represent the mean ± standard deviation of six replicates. The different letters followed the values in the same column indicate significant difference between the treatments at p < 0.05. The same letters followed the values in the same column indicate no significant difference between the treatments at p < 0.05.

50 ␮M Cd were greater than those of control plants; the increase of Zn-treated root biomass was statistically significant (Fig. 1A, B). Notably, fungal inoculation significantly increased shoot and root biomasses of seedlings grown with 500 ␮M Zn or 50 ␮M Cd; the respective increases were 37% and 28% for seedlings grown with 500 ␮M Zn, and 33% and 31% for seedlings grown with 50 ␮M Cd (Fig. 1A, B).

3.2. Effects of fungal inoculation in hydroponic cultures

3.2.2. Root system Corresponding to the increase of root biomass, fungal inoculation led to greater root systems. Root length, number of root tips, and root surface area of the inoculated seedlings treated with or without heavy metals were generally significantly greater than those of the correspondingly noninoculated seedlings, except the root length of inoculated seedlings without heavy metal treatments (Table 2).

3.2.1. Biomass Fifteen days after inoculation, the shoot and root biomasses of hydroponic seedlings inoculated with the SaCS12 fungus were already significantly greater than those of control plants (Fig. 1A, B). Fifteen days after heavy metal treatment, the shoot and root biomasses of hydroponic seedlings grown with 500 ␮M Zn or

3.2.3. Chlorophyll, N and Mg concentrations Fifteen days after treatments, leaf chlorophyll concentrations of seedlings treated with Zn or Cd were higher than those of control seedlings; the difference between Zn-treated and control seedlings was significant. Fifteen days after inoculation, the chlorophyll concentrations of the inoculated seedlings treated with or without

Fig. 1. Biomasses of hydroponic S. alfredii seedlings treated with 500 ␮M Zn or 50 ␮M Cd, and/or F. oxysporum inoculation. The different letters on the error bars indicate significant difference of shoot or root biomass between treatments at p < 0.05. The same letters on the error bars indicate no significant difference between treatments at p < 0.05.

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Table 3 Chlorophyll, N and Mg concentrations of hydroponic S. alfredii seedlings treated with 500 ␮M Zn or 50 ␮M Cd, and/or F. oxysporum inoculation. Treatments

Chlorophyll concentration (mg g−1 fresh weight)

N concentration(mg kg−1 )

Shoot

Mg concentration (mg kg−1 )

Root

Control Inoculation Zn Zn + Inoculation

0.49 0.59 0.56 0.75

± ± ± ±

0.03c 0.06b 0.03b 0.04a

10,664 16,718 13,141 22,614

± ± ± ±

Control Inoculation Cd Cd + Inoculation

0.47 0.61 0.53 0.69

± ± ± ±

0.05c 0.04b 0.04c 0.05a

11,567 ± 757c 15,240 ± 632b 13,461 ± 1303bc 21,521 ± 181a

1418c 1413b 523bc 3250a

Shoot

Root

10,370 14,954 12,304 20,063

± ± ± ±

1349c 1263b 421bc 1337a

5159 6068 5981 7142

± ± ± ±

348c 399b 392bc 592a

1769 2287 2182 2906

± ± ± ±

78c 313b 295bc 300a

9560 11,964 11,768 15,543

± ± ± ±

658c 946b 934bc 1905a

5235 6183 6088 7294

± ± ± ±

524c 423b 410bc 534a

1721 2166 2024 2652

± ± ± ±

70c 250b 219bc 256a

Values represent the mean ± standard deviation of six replicates. The different letters followed the values in the same column indicate significant difference between the treatments at p < 0.05.The same letters followed the values in the same column indicate no significant difference between the treatments at p < 0.05. Table 4 Zn or Cd mass and concentration in hydroponic S. alfredii seedlings treated with 500 ␮M Zn or 50 ␮M Cd, and/or F. oxysporum inoculation. Treatments

Zn concentration (mg kg−1 )

Zn mass (10−3 mg plant−1 )

Shoots Control Inoculation Zn Zn + Inoculation Treatments

1960 2134 13,219 17,431

± ± ± ±

Roots 252c 246c 714b 967a

± ± ± ±

Shoots 21b 30b 440a 458a

Cd concentration (mg kg−1 ) Shoots

Control Inoculation Cd Cd + Inoculation

392 412 3681 3431

45 56 3636 4761

± ± ± ±

± ± ± ±

Roots 28d 56c 173b 480a

17 22 198 238

± ± ± ±

3b 3b 38a 47a

Cd mass (10−3 mg plant−1 ) Roots

13c 8c 222b 242a

279 425 2318 4197

7.8 8.1 544.3 558.2

Shoots ± ± ± ±

1.4b 2.1b 50.1a 90.1a

6.9 8.2 600.7 1034.1

Roots ± ± ± ±

1.2c 2.1c 35.9b 158.8a

0.3 0.4 26.0 34.9

± ± ± ±

0.1c 0.1c 4.9b 2.8a

Values represent the mean ± standard deviation of six replicates. The different letters followed the values in the same column indicate significant difference between the treatments at p < 0.05. The same letters followed the values in the same column indicate no significant difference between the treatments at p < 0.05.

heavy metals were significantly higher than those of the correspondingly noninoculated seedlings (Table 3). Consistently, the concentrations of shoot N and Mg, the components of chlorophyll, were significantly higher in the inoculated seedlings treated with or without heavy metals compared with those of the noninoculated seedlings (Table 3). Likewise, the concentrations of root N and Mg in the inoculated seedlings were significantly higher than those of the correspondingly noninoculated seedlings (Table 3). 3.2.4. Heavy metal uptake Control seedlings absorbed Zn from the nutrient solution containing 1 ␮M Zn. In the hydroponic culture containing 500 ␮M Zn, seedlings hyperaccumulated Zn (1.32%, w/w) in shoots with a TF of 3.6 ± 0.2; fungal inoculation did not changed the Zn concentration and mass in roots but significantly increased the Zn concentration (32%) and mass (81%) in shoots (Table 4) and the TF to 5.1 ± 0.5. Control seedlings grown in the Cd-free nutrient solution contained a low concentration of Cd originated from the last culture. In the hydroponic culture containing 50 ␮M Cd, seedlings hyperaccumulated Cd (0.36%, w/w) in shoots with a TF of 6.7 ± 0.3; fungal inoculation did not changed the Cd concentration in roots but significantly increased the Cd mass (35%) in roots, the Cd concentration (31%) and mass (72%)in shoots (Table 4) and the TF to 8.6 ± 1.1. 3.3. Effects of fungal inoculation in multi-metal contaminated soils 3.3.1. Soil metal availability Physicochemical properties of the mined soil and contaminated paddy soil are presented in Table 5. The concentrations of available

N, P, K, Fe, Zn and Cu in the mined soil were lower than those in the paddy soil, among which the available Cu concentration was much lower than that in the paddy soil; the concentrations of available Mg, Cd and Pb in the mined soil were higher than those in the paddy Table 5 Physicochemical properties of the multi-metal contaminated soils used in this study. Soil parameters

Mined soil

Paddy soil

Sand (2–0.02 mm) % Silt (0.02–0.002 mm) % Clay (<0.002 mm) %

82 ± 0.84 15.0 ± 0.22 3.0 ± 0.06

70 ± 0.72 27.4 ± 0.38 2.6 ± 0.05

Texture pH (soil: water 1:2.5) Organic matter (g kg−1 ) Cation exchange capacity (cmolc kg−1 ) Total N (mg kg−1 ) Total P (mg kg−1 ) Total K (mg kg−1 ) Total Mg (mg kg−1 ) Total Fe (mg kg−1 ) Total Zn (mg kg−1 ) Total Cd (mg kg−1 ) Total Pb (mg kg−1 ) Total Cu (mg kg−1 ) Available N (mg kg−1 ) Available P (mg kg−1 ) Available K (mg kg−1 ) Available Mg (mg kg−1 ) Available Fe (mg kg−1 ) Available Zn (mg kg−1 ) Available Cd (mg kg−1 ) Available Pb (mg kg−1 ) Available Cu (mg kg−1 )

Sandy loam 7.32 ± 0.11 15.82 ± 1.2 8.63 ± 0.57 456.11 818 7169 2530 36,257 3699 41.70 8546 150 100 1.56 81.61 350 57.22 986 12.75 320.0 35.42

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

20.33 47.31 540 157 2098 213 2.1 499 8 7 0.08 4.13 18 2.99 55 0.64 18 1.88

Sandy loam 7.81 ± 0.13 28.62 ± 1.6 10.21 ± 0.54 741.32 885 10,801 4061 11,344 3078 10.32 232 1391 130 3.74 90.64 279 83.45 1903 5.07 25.32 821.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

37.09 46.45 765 246 685 221 0.52 14 78 8 0.21 4.53 14 4.17 114 0.28 1.44 56

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Table 6 Soil metal availabilities after S. alfredii plantation and F. oxysporum inoculation. Soil types

Treatments

Zn (mg kg−1 )

Mined soil

Control Inoculation S. alfredii S. alfredii + Inoculation

1001 1063 1216 1480

Soil types Paddy soil

Treatments Control Inoculation S. alfredii S. alfredii + Inoculation

Zn (mg kg−1 ) 1906 ± 105a 1934 ± 92a 1980 ± 194a 1922 ± 191a

± ± ± ±

66c 103c 78b 102a

Cd (mg kg−1 ) 12.21 12.63 13.38 17.10

± ± ± ±

1.46b 0.51b 1.53b 1.39a

Cd (mg kg−1 ) 4.91 ± 0.34ab 5.03 ± 0.14a 4.53 ± 0.30bc 4.43 ± 0.27c

Pb (mg kg−1 ) 309 335 419 544

± ± ± ±

31c 20c 30b 36a

Pb (mg kg−1 ) 26.60 ± 0.43c 27.38 ± 1.41c 38.24 ± 3.21b 54.24 ± 6.81a

Cu (mg kg−1 ) 36.74 35.70 35.05 33.92

± ± ± ±

3.30a 4.02a 2.79a 3.67a

Cu (mg kg−1 ) 838 ± 25ab 873 ± 23a 840 ± 24ab 819 ± 29b

Values represent the mean ± standard deviation of six replicates. The different letters followed the values in the same column indicate significant difference between treatments in the same soil at p < 0.05. The same letters followed the values indicate no significant difference between the treatments at p < 0.05.

soil, among which the available Pb concentration was much higher than that in the paddy soil. At five months after plantation, S. alfredii plants significantly increased the concentrations of available Zn and Pb in the mined soil and Pb in the paddy soil compared with those of control soils without plantation and fungal inoculation (Table 6). At five months after inoculation, the SaCS12 fungus did not change the concentrations of available heavy meals in both soils without plantation (Table 6). S. alfredii plantation combining with fungal inoculation significantly increased the concentrations of available Zn, Cd and Pb in the mined soil and Pb in the paddy soil compared with those in soils without inoculation; the plantation and inoculation did not change the concentrations of available Cu in the mined soil and Zn, Cd and Cu in the paddy soil (Table 6) in which the originally available Zn, Cd and Cu were at very high rates of 61.8%, 49.1% and 59.0%, respectively. 3.3.2. Biomass One month after plantation to soils and inoculation with the SaCS12 fungus, the shoots of inoculated plants showed greater sizes compared with those of the noninoculated plants (Fig.S2). At five months after transplantation and inoculation, the biomasses of harvested shoots and roots from inoculated plants grown in the mined soil were respectively 1.57-fold and 1.70-fold of those from noninoculated plants; the biomasses of harvested shoots and roots

from inoculated plants grown in the paddy soil were respectively 1.24-fold and 1.41-fold of those from noninoculated plants (Fig. 2A, B). The biomasses of shoots and roots from noninoculated plants grown in the paddy soil were higher than those grown in the mined soil; the difference between root biomasses was significant. The biomasses from inoculated plants grown in the two types of soils did not shown significant difference (Fig. 2A, B). Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2012.06.013.

3.3.3. Heavy metal uptake and phytoextraction from soils S. alfredii plants grown in both type of soils accumulated most of Zn (98.9% and 97.6%), Cd (99.6% and 97.9%), Pb (91.3% and 87.3%), and Cu (88.9% and 65.0%) masses in shoots, accumulated high concentrations of Zn and Cd in shoots and high concentrations of Pb and Cu in roots(Table 7). The concentrations of Zn, Pb, and Cu in roots and shoots were seemingly positively correlated with the available metal concentrations in soils before plantation (Tables 5 and 7). Plants grown in the paddy soil accumulated a higher concentration of Zn in roots but a lower concentration of Zn in shoots, a similar concentration of Cd in roots but a lower concentration of Cd in shoots compared with those in the mined soil (Table 7); they thus had lower TFs for Zn and Cd compared with those in the mined soil (Table 8).

Fig. 2. Effects of F. oxysporum inoculation on biomass of S. alfredii plants grown in multi-metal contaminated soils. Each column represents the mean of six replicates; error bars represent standard deviations. The different letters on the error bars indicate significant difference between treatments in the same soil at p < 0.05. *indicates significant difference between two types of soils.

1940a 11a* 54b* 44b* ± ± ± ± 8290 83 247 322 Zn Cd Pb Cu Paddy soil

Values represent the mean ± standard deviation of six replicates. The different letters followed the values indicate significant difference between control and inoculation treatments in the same soil at p < 0.05. The same letters followed the values indicate no significant difference between control and inoculation treatments at p < 0.05. *indicates significant difference between two types of soils.

1.33a* 0.02a 0.12a* 0.85a* ± ± ± ± 5.84 0.05 1.08 5.13 18a* 0.39a* 1.11a* 2.06a* ± ± ± ± ± ± ± ±

1616a* 13a* 56a* 79a*

4042 34 725 3426

± ± ± ±

705a 8a 81b 532b

4807 41 901 4254

± ± ± ±

638a 10a 83a 247a

139 ± 33b 1.39 ± 0.22b* 4.11 ± 0.44b* 5.39 ± 0.58b*

200 2.16 7.68 9.66

3.42 ± 0.36b* 0.03 ± 0.01a 0.62 ± 0.09b* 2.93 ± 0.55b*

0.36a 0.01a 0.55a 0.01a ± ± ± ± 3.05 0.04 4.34 0.14 ± ± ± ± 1.52 0.02 1.89 0.07 41a 1.39a 8a 0.19a ± ± ± ± ± ± ± ±

Control

136 5.60 20 0.57 197a 7a 160a 14a ± ± ± ±

Inoculation

2925 33 4158 138 413a 4a 297b 15a ± ± ± ± 2543 35 3098 116 1431a 56a 351a 9a ± ± ± ±

Inoculation

15,315 617 1624 41 962b 76b 443a 9a 10,089 418 1491 42 Zn Cd Pb Cu Mined soil

± ± ± ±

9679 103 369 461

Control Control

333 13.42 35 0.91

Control

Shoot Root Shoot

14b 0.48b 5b 0.14b

Metal mass (mg pot−1 ) Metal concentration (mg kg−1 ) Heavy metal Soil type

Table 7 Metal mass and concentrations in S. alfredii plants grown in multi-metal contaminated soils with and without F. oxysporum inoculation.

Inoculation

Root

0.16b 0.01a 0.38b 0.02b

Inoculation

X. Zhang et al. / Journal of Hazardous Materials 229–230 (2012) 361–370

367

After plantation with and without fungal inoculation, the available metals in the soils were positively correlated with the Zn, Cd and Pb concentrations in the plants grown in the mined soil and Zn and Pb concentrations in the plants grown in the paddy soil. Metal concentrations in the accumulating organs determined the correlation (Table 9). In the mined soil fungal inoculation significantly increased Pb concentration in roots, Zn and Cd concentrations in shoots (Table 7), and Zn- and Cd-TFs of plants (Table 8). In the paddy soil fungal inoculation significantly increased Pb and Cu concentrations in roots and shoots (Table 7) but not the corresponding TFs of plants (Table 8). Correspondingly, fungal inoculation significantly increased Zn-, Cd- and Pb-BAFs of plants grown in the mined soil and Pb- and Cu-BAFs of plants grown in the paddy soil (Table 8). Fungal inoculation generally increased metal masses in shoots and roots (Table 7), consequently, increased all of the metal PRs of the plants grown in both soils (Table 8); fungal inoculation increased PR of Zn, Cd, Pb, and Cu at 144%, 139%, 84%, and 63% in the mined soil, at 44%, 55%, 85%, and 77% in the paddy soil, respectively.

4. Discussion Previous studies have demonstrated that the hyperaccumulating ecotype S. alfredii plants are able to hyperaccumulate Zn and Cd and accumulate Pb in shoots grown in hydroponic cultures and natural soils at Pb/Zn mined areas [10–12,32–35]. In response to high levels of external heavy metals, S. alfredii hyperaccumulator could enhance root exudation to mobilize soil metal elements, enlarge root systems and surface areas to uptake metals, and increase metal root-to-shoot translocation [36,37]. Here, our results clearly showed that a Fusarium fungus originally associated with the S. alfredii hyperaccumulator grown in the Pb/Zn mined soil could further enhance these host responses, leading to significantly greater host biomass and metal accumulation and together a bioaugmentation of phytoextraction. The results from the hydroponic systems showed the potentials for the Fusarium fungus to promote plant growth and metal extraction and also showed a clue for the fungus-induced synergistic promotion. The fungal hyphal networks assist root uptake of nutrients including N and Mg, the components of chlorophyll, and thus increase shoot synthesis of chlorophylls and photosynthetic efficiency; in turn, more assimilates support more root exudation and larger root system and surface areas, and thus more bioavailable metals, more root accessible areas, and metal uptake. The S. alfredii-mediated increase of soil metal availability appeared to be varied with metal species, concentration, and other soil physicochemical properties. Seemingly, the S. alfredii-mediated increase of Zn, Cd and Pb availabilities in the Pb/Zn mined soil is related to the plant ability to accumulate Zn, Cd, and Pb. Neither S. alfredii plants showed effects on Cu availability in the multi-metal contaminated soils, nor did Cu concentrations in plants show correlation with the available Cu concentrations in soils. However, the S. alfredii plants showed a tolerance to the high Cu concentration in the paddy soil. They accumulated respective 7.67- and 29.5- fold concentrations of Cu in shoots and roots in the paddy soil compared with those in the mined soil and did not show growth disadvantages. On the other hand, the high level of soluble Cu did not inhibit root uptake of Zn, Cd, and Pb but seemed inhibit the root-to-shoot translocation of Zn and Cd. Correspondingly, a previous study showed that the hyperaccumulating ecotype S. alfredii accumulated increased concentrations of Cu in roots and decreased concentrations of Cu in leaves when the external Cd was higher than 50 ␮M [11]. The potential antagonistic interactions between Zn/Cd and Cu translocation needs further investigations.

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Table 8 Translocation factors (TF), bioaccumulation factors (BAF) and phytoextraction rates (PR) for heavy metals by S. alfredii plants grown in multi-metal contaminated soils with and without F. oxysporum inoculation. Soil types

Treatments

Zn

Cd

TF Mined soil Paddy soil

Soil types

Control Inoculation Control Inoculation Treatments

4.01 5.24 2.06 2.07

BAF ± ± ± ±

0.37b 0.38a 0.37a* 0.63a*

Paddy soil

Control Inoculation Control Inoculation

± ± ± ±

0.26b 0.39a 0.63a 0.53a

3.72 9.09 4.63 6.69

TF

± ± ± ±

0.37b 1.09a 1.07b 0.53a

Pb

0.48 0.39 0.34 0.40

BAF

12.02 19.24 2.52 2.70

± ± ± ±

1.01b 3.68a 0.70a* 1.00a*

10.02 14.81 8.03 9.98

PR (%) ± ± ± ±

1.83b 1.34a 1.11a 1.33a

± ± ± ±

0.10a 0.10a 0.38b 0.18a

13.47 32.26 13.80 21.40

± ± ± ±

1.14b 3.30a 2.11b 3.65a

Cu

TF Mined soil

2.73 4.14 2.69 3.14

PR (%)

BAF ± ± ± ±

0.11a 0.09a 0.04a 0.04a

0.36 0.49 3.13 3.89

PR (%) ± ± ± ±

0.04b 0.02a 0.35b 0.36a

0.25 0.46 2.04 3.77

± ± ± ±

TF 0.06b 0.09a 0.23b 0.50a

0.37 0.31 0.10 0.11

BAF ± ± ± ±

0.07a 0.10a 0.03a* 0.02a*

0.77 0.92 2.46 3.06

PR (%) 0.43 0.70 0.60 1.06

± ± ± ±

0.10b 0.12a 0.08b 0.16a

BAF for Zn or Cd is the ratio of metal concentration in shoots to metal concentration in soils; BAF for Pb or Cu is the ratio of metal concentration in roots to metal concentration in soils. PR for the heavy metals is the ratio (percentage) of the metal mass in plants to that in the soils before plantation. Values represent the mean ± standard deviation of six replicates. The different letters followed the values indicate significant difference between control and inoculation treatments in the same soil at p < 0.05. The same letters followed the values indicate no significant difference between control and inoculation treatments at p < 0.05. *indicates significant difference between TFs of plants grown in two types of soils.

The nonpathogenic F. oxysporum fungus-enhanced phytoextraction was more pronounced for the S. alfredii hyperaccumulator grown in the Pb/Zn mined soil than in the contaminated paddy soil. It is known that the efficiency of microorganism-assisted phytoextraction depends on many factors including metal species, concentration, and competition, soil physicochemical properties, plant physiology, root architecture, and their growth conditions, and the interactions between plants and microbes [3]. Seemingly, the S. alfredii hyperaccumulator and its fungal partner have been adapted to the mined soil with high levels of Zn and Pb and low to moderate levels of Cd. The low-biomass hyperaccumulators are disadvantageous for remediation of a large area of polluted soils compared with using high-biomass plants [38,39]. Our results showed a two-fold phytoextraction of multi-metals from the mined soil by the Fusarium fungus-assisted S. alfredii hyperaccumulator and thus a greater potential for field application of the fungus-hyperaccumulator cooperated phytoextraction. The Fusarium fungus-enhanced root growth and development and metal uptake are reminiscent of AMF-assisted phytoextration [3,8]. However, a recent study showed that AMF colonization of S. alfredii were negatively correlated with soil bioavailable Pb and Cu and not significantly correlated with S. alfredii accumulation of Zn, Cd, Pb, Cu, and As [21]. Therefore, this Fusarium fungal partner presents a promising alternative to enhance phytoextraction by the S. alfredii hyperaccumulator.

AMF are the most common and widespread symbiotic fungi to the terrestrial plants. However, they are obligate biotrophs and cannot be cultured without the plants [40]. Therefore, the large scale application of AMF inoculation to assist the phytoextraction in the fields is limited. Moreover, most of the known hyperaccumulators belong to the Brassicaceae familiy, such as Thlaspi caerulescens, Brassica juncea and Arabidopsis halleri, and are typically non-mycorrhized plants [3]. The Fusarium fungus used in this study is easy to be mass-produced. This study thus highlights the potentials for wide use of culturable indigenous fungi associated with hyperaccumulators and accumulators other than AMF to enhance phytoextraction. F. oxysporum fungi are common fungi found in soils worldwide and are often in association with plant roots [41]. Although F. oxysporum fungi have been studied primarily because they cause diseases of economically important plant hosts, nonpathogenic F. oxysporum fungi are predominantly associated with plants [41]. Nonpathogenic F. oxysporum fungi have been studied mainly becauseof their potential to control pathogenic F. oxysporum fungi [42,43]. Diverse biochemical and ecological activities of F. oxysporum fungi are being explored [44–46]. A recent study showed that petroleum-resistant F. oxysporum fungi and other Fusarium fungi could efficiently remediate petroleum-polluted soils together with plants [47]. Our study for the first time showed that a metalresistant nonpathogenic F. oxysporum fungus promoted metal phytoextraction by a hyperaccumulator.

Table 9 Pearson correlation coefficients between concentrations of heavy metals accumulated in plants and concentrations of heavy metals available in soils after plantation with and without fungal inoculation. Soil types

Metals

Roots

Shoots

Plants

Mined soil

Zn Cd Pb Cu

0.841* N.S. 0.960** N.S.

0.986** 0.952** N.S. N.S.

0.990** 0.953** 0.968** N.S.

Paddy soil

Zn Cd Pb Cu

N.S. 0.843* 0.952** N.S.

0.815* N.S. 0.969** N.S.

0.780* N.S. 0.965** N.S.

**p < 0.01; *p < 0.05; N.S.: no significant correlation.

Pearson correlation coefficients

X. Zhang et al. / Journal of Hazardous Materials 229–230 (2012) 361–370

5. Conclusion This study showed that a culturable indigenous Fusarium oxysporum fungus isolated from the Zn/Cd co-hyperaccumulator S. alfredii could enhance host-mediated soil metal mobilization, metal uptake or translocation, enlarge root systems, increase chlorophyll synthesis and plant biomass, leading to approximately 2-fold phytoextraction of multi-metals from the Pb/Zn mined soil. Therefore, these results showed an enhanced application potential for the S. alfredii hyperaccumulator. This is the first report that showed an indigenous culturable Fusarium fungus other than mycorrhizal fungi to enhance phytoextraction by hyperaccumulators. This report showed a new avenue of microorganism-assisted phytoextraction for hyperaccumulators that are mainly nonmycorrhized Brassicaceae plants. Moreover, this report showed a new function and application potential for the worldwide distributed soil and plant-associated nonpathogenic F. oxysproum fungi.

Acknowledgments This work was supported by the project from Natural Science Foundation of China (21177107), the key project from the Ministry of Education, China (310003), and the “863” project from the Ministry of Science and Technology of China (2009AA06Z316).

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