Arbuscular mycorrhizal fungi alleviate the heavy metal

Science of the Total Environment 628–629 (2018) 282–290

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Arbuscular mycorrhizal fungi alleviate the heavy metal toxicity on sunflower (Helianthus annuus L.) plants cultivated on a heavily contaminated field soil at a WEEE-recycling site Yu Zhang a,b, Junli Hu a,⁎, Jianfeng Bai b,⁎, Junhua Wang a, Rui Yin a, Jingwei Wang b, Xiangui Lin a a State Key Laboratory of Soil and Sustainable Agriculture, Joint Open Laboratory of Soil and the Environment, Hong Kong Baptist University & Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China b Shanghai Collaborative Innovation Centre for WEEE Recycling, WEEE Research Centre of Shanghai Polytechnic University, Shanghai 201209, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• WEEE-recycling induced serious heavy metal (HM) contamination on soil and sunfower. • AM fungal inoculation (+M) enhanced both P absorbtion and plant growth of sunfower. • +M significantly decreased HM levels in the shoot, but not the root, of sunfower. • +M increased leaf catalase activity and reduced HM toxicity on thylakoid structure. • +M promoted HM uptakes, suggesting its potential application for phytoextraction.

a r t i c l e

i n f o

Article history: Received 6 December 2017 Received in revised form 31 January 2018 Accepted 31 January 2018 Available online xxxx Editor: Xinbin Feng Keywords: Catalase Funneliformis caledonium Funneliformis mosseae P acquisition Phytoextraction Transmission electron microscope

a b s t r a c t An 8-week pot experiment was conducted to investigate the growth and responses of sunflower (Helianthus annuus L.) to arbuscular mycorrhizal (AM) fungal inoculations on a heavily heavy metal (HM)-contaminated (H) soil and a lightly HM-enriched (L) soil, both of which were collected from a waste electrical and electronic equipment (WEEE)-recycling site. Compared with the L soil, the H soil induced significantly larger (P b 0.05) concentrations of Cd, Cu, Pb, Cr, Zn and Ni in sunflower (except for root Cr and shoot Ni), which impaired the thylakoid lamellar folds in leaves. The biomasses and P concentrations of shoots and roots, as well as the total P acquisitions per pot were all significantly decreased (P b 0.05). Both Funneliformis mosseae (Fm) and F. caledonium (Fc) inoculation significantly increased (P b 0.05) root mycorrhizal colonization. For the L soil, AM fungal inoculations had no significant effects on the soil-plant system, except for a decrease of soil pH and increases of soil available P and DTPA-extractable Zn concentrations with the Fm-inoculated treatment. For the H soil, however, AM fungal inoculations significantly increased (P b 0.05) the biomasses and P concentrations of shoots and roots, as well as the total P acquisitions per pot, and significantly reduced (P b 0.05) the concentrations of HMs in shoots (except for Cu and Pb with Fm- and Fc- inoculated treatments, respectively) and alleviated the toxicity symptoms of HMs in thylakoid structure of leaves. AM fungal inoculations in the H soil also

⁎ Corresponding authors at: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, China; Shanghai Collaborative Innovation Centre for WEEE Recycling, WEEE Research Centre of Shanghai Polytechnic University, China. E-mail addresses: [email protected] (J. Hu), [email protected] (J. Bai).

https://doi.org/10.1016/j.scitotenv.2018.01.331 0048-9697/© 2018 Elsevier B.V. All rights reserved.

Y. Zhang et al. / Science of the Total Environment 628–629 (2018) 282–290

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significantly increased (P b 0.05) the shoot uptake of HMs (except for Cr), and tended to decrease the total concentrations of HMs in soils. This suggests the potential application of AM fungi for both reducing HM stress and promoting phytoextraction of HM-contaminated soils caused by WEEE recycling. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Waste electrical and electronic equipment (WEEE) refer to end-oflife electronic products, including television sets, washing machines, air conditioners, computers, mobile phones, and others, which are composed of sophisticated blends of plastics and metals, among other materials (Xu et al., 2015). WEEE contain reusable precious metal resources, such as Au, Ag, Pd and Pt (Zhao et al., 2015). Due to the cheap labor cost and the weak legislation system, a number of WEEE-recycling workshops have been assembled in some cities of developing countries, such as Guiyu and Taizhou of China, Delhi and Bengaluru of India, Lagos of Nigeria, and Accra of Ghana (Xu et al., 2015). In these areas, WEEE are recycled by local villagers using primitive disassembly methods, without appropriate facilities to prevent environmental pollution (Damrongsiri et al., 2016). Therefore, tens of millions of tons of WEEE-recycling residues were dumped in workshops, yards, roadsides, open fields, wastelands, irrigation canals, ponds, rivers and riversides every year (Huo et al., 2007). Hazardous chemicals, such as heavy metals (HMs), can be released from the disposal or recycling processes of WEEE, polluting the environment (Zhang et al., 2014). In fact, extensive research has reported heavy pollutions of HMs (such as Cd, Cu, Pb, Cr, Zn and Ni) in soils (Tang et al., 2010; Wu et al., 2015; Zhao et al., 2015), vegetables (Luo et al., 2011), and crops (Fu et al., 2008) in neighboring fields due to WEEE recycling. Large concentrations of HMs in the soil may inhibit seed germination and/or increase the HM accumulation risks of plants, and even influence nutrient metabolism or reduce the photosynthetic and growth rates when the accumulation exceeds a certain threshold (Andrade et al., 2010; Subba et al., 2014; Gill et al., 2015; Gupta et al., 2017). In fact, damage to plant organs due to large concentrations of HMs has been widely observed, such as the thinning of the cell wall, the formation of intercellular spaces, and amoeboid protrusions and folds, and the appearance of immature nucleus and ruptured thylakoid membranes (Kaur et al., 2013; Gill et al., 2015). In addition, plants treated with large concentrations of HMs usually show a significant reduction of chlorophyll and carotenoid contents (Subba et al., 2014), as well as the generation of redox imbalance (Gupta et al., 2017). Arbuscular mycorrhizal (AM) fungi can form symbiotic associations with most terrestrial plant species in a wide range of soils (Hassan et al., 2013), and can improve plant performance through increased defenses against environmental stresses, both biotic and abiotic, such as drought, salinity, and HM toxicity (Ferrol et al., 2016). In the extreme environment with large concentrations of various HMs, AM fungi can survive (Sanchez-Castro et al., 2017) and are known to have beneficial effects on host plants. On the one hand, AM fungi can make a considerable contribution to nutrient (notably P) uptake to promote plant growth (Smith and Read, 2008). On the other hand, AM fungi can alleviate HM toxicity on plants in polluted soil by the reduction of HM acquisitions (Hu et al., 2014), the biological dilution of HMs (Hu et al., 2013), and the decrease of oxidative stress (Neagoe et al., 2014). Consequently, there may be a potential of using AM fungi to enhance plant resistance to environmental stress caused by HM contamination. For example, Funneliformis mosseae (Fm) could reduce the concentrations of Pb, Cd and As of horse tamarind (Leucaena leucocephala (Lam.) de Wit) (Zhan et al., 2016), while F. caledonium (Fc) could decrease shoot Cd, Cu, Pb, and Zn concentrations of maize (Zea mays L.) (Wang et al., 2007). However, different AM fungal species may induce differential effects on the absorption of elements by the same plant. For example, there was a significant difference in Cu-sorption capacity between the extraradical mycelium of Fc and Fm (Gonzalez-Chavez et al., 2002),

and inoculation with Fc and Fm could induce differential U and Cd absorption by Chinese brake fern (Pteris vittata L.) and Alfred stonecrop (Sedum alfredii Hance), respectively (Chen et al., 2006; Hu et al., 2013). To be a potential material for phytoremediation that can survive in heavily contaminated soils with various HMs, ideal plants should have a considerable capacity for HM tolerance and a fast growth rate with a large biomass. Sunflower (Helianthus annuus L.), a food, oil and fuel bioenergy plant species (Fozia et al., 2008), grows in a wide range of soils throughout the world (Forte and Mutiti, 2017). Its stem can grow as high as 3 m tall with the flower head reaching up to 30 cm within 2 or 3 months (Adesodun et al., 2009; Jadia and Fulekar, 2008), and it can accumulate relatively large concentrations of Cd (Lopes Júnior et al., 2014), Cu (Forte and Mutiti, 2017), Pb (Batista et al., 2017), Cr (Cutright et al., 2010), Zn (Adesodun et al., 2009), and Ni (Shaheen and Rinklebe, 2015). However, most former studies focused on the growth of sunflower on mild or moderate contaminated (only by one to two kinds of HMs) soils (de Andrade et al., 2008; Fozia et al., 2008; Adesodun et al., 2009; Jarrah et al., 2014). There is limited information available for the role of AM fungi on plant growth in heavily contaminated soil with various HMs, such as caused by WEEE recycling. Thus, it was hypothesized that AM fungal inoculation could alleviate the stress of HMs on plants via decreasing HM concentrations, and different AM fungal species may induce differential effects on the absorption of various HMs by sunflower. The objective of this experiment was therefore to investigate the growth of sunflower on a heavily HM-contaminated (H) soil and a lightly HM-enriched (L) soil, both of which were collected from a WEEE-recycling site, and the plant's responses to the inoculations of two different AM fungal species. This study may contribute to developing application strategies of AM fungi to reduce HM stress and promote plant growth in HM-contaminated soils around WEEErecycling sites. 2. Materials and methods 2.1. Mycorrhizal inoculum Two AM fungal inocula were used in this experiment. Funneliformis mosseae (Nicol. & Gerd.) Gerd. & Trappe M47V (Fm) was obtained from the International Bank for Glomeromycota (IBG), France. F. caledonium (Nicolson & Gerd.) Walker & Schüßler 90036 (Fc) was deposited at the Institute of Soil Science, Chinese Academy of Sciences, China. The two AM fungal inocula and the non-mycorrhizal inoculum were prepared using the same substrate under the same conditions. After plant harvesting, all inocula were air-dried and passed through a 2-mm sieve. 2.2. Soil preparation Two soil samples were collected from a residue-dumping (RD) site (28°30′47″N, 121°24′16″E), which was used for the dumping of WEEE-recycling residues, and a neighboring field (NF) site (28°30′42″ N, 121°24′21″E), which was an arable agricultural land 210 m far away from the RD site, in Taizhou City, Zhejiang Province, one of the three major WEEE-recycling bases in China. The soil samples were airdried, ground with a wooden pestle, and homogenized by sieving through a 5-mm sieve. Subsamples of soils were separately sieved through a 2 mm sieve for analyzing soil properties. Soil pH (soil: deionized water = 1:5) was measured with a pH meter (Thermo Scientific Orion Star A211, USA). Soil available P was extracted with sodium

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bicarbonate (NaHCO3) and determined by the molybdenum-blue method (Olsen et al., 1954). Soil total Cd, Cu, Pb, Cr, Zn, Ni and P concentrations were determined using HPLC-ICP-MS (Agilent 7700×, USA) after the subsamples (0.1 g) were sieved through a 0.149 mm sieve and digested with an acid mixture (8 mL nitric acid, 1 mL perchloric acid and 4 mL hydrofluoric acid). Both standard reference materials [GBW07457 (GSS-28) were obtained from the Institute of Geophysical and Geochemical Exploration, Chinese Academy of Geological Sciences (IGGE)], and blanks were included for quality assurance. The recovery rates of soil total Cd, Cu, Pb, Cr, Zn, and Ni were between 94.3% and 107.8%. Soil DTPA-extractable concentrations [0.1 M triethanolamine (TEA), 0.01 M CaCl2 and 0.005 M diethylenetriaminepentaacetic acid (DTPA), pH 7.3, solution: soil = 2:1, extraction for 2 h (Baker and Amacher, 1982)] of Cd, Cu, Pb, Zn and Ni were measured using ICPOES (PerkinElmer Optima8000, USA) and were expressed as an ovendried soil weight for correcting the water content in the soil (105 °C, 7 h). This method is not suitable for the determination of Cr, so the corresponding data were not shown. The soil collected from the NF site contained 30.2 g kg−1 organic C, 3.07 g kg−1 of total P and 65.2 mg kg−1 of available P, and had a pH (H2O) of 5.52, and the concentrations of Pb, Cr, and Zn all met the requirements set by China with pH b 6.5 (SEPAC, 1995), while the remaining HMs (Cd, Cu, and Ni) lightly exceeded the national standard (Table 1). The soil collected from the RD site contained 94.3 g kg−1 organic C, 1.00 g kg−1 of total P and 0.84 mg kg−1 of available P, and had a pH (H2O) of 8.06, and HM concentrations all greatly exceeded the national standard with pH N 7.5. Therefore, the soils collected from the RD and NF sites were heavily HM-contaminated (H) soil and lightly HM-enriched (L) soil, respectively.

2.3. Pot experiment Three treatments were established for each test soil (L and H): noninoculated control, inoculation with Fm or Fc. Soil samples, 4 kg each, were put in a polyvinyl chloride pot (23.5 cm diameter × 18 cm depth), followed by a thin layer (250 g) of AM inocula or nonmycorrhizal inoculum, four sunflower seeds and 0.5 kg of casing soils. Sunflower seedlings were thinned to two after germination. Pots were randomly arranged with four replicates per treatment. Plants were watered to maintain soil moisture at approximately 80% of the waterholding capacity by adding tap water. Every two weeks (with a total of three times), 200 ml of 50% Hoagland nutrient solution was applied

to each pot. After growing for 8 weeks, all plants were harvested, and soil samples were also collected.

2.4. Mycorrhizal colonization and plant analysis To investigate the effects of heavy contamination of HMs on the leaves of sunflower and their responses to AM fungal inoculations, the control of the L soil, and the control and one of the AM-inoculated treatments of the H soil, were selected for antioxidant catalase (CAT) activity determination and transmission electron microscope (TEM) observation. Before harvest, the subsamples of fresh leaves at the same location of each plant were collected, washed, and weighed. The activity of antioxidant CAT was measured using a CAT ELISA Kit (DG, Beijing, China). The leaf tissue samples used for TEM observation were prefixed in 4% glutaraldehyde, washed in 0.1 mol L−1 pH 7.0 phosphate buffer, postfixed in 1% osmium tetroxide for 2 h, dehydrated in a graded series of ethanol, embedded in epoxy resin (STON 812), sectioned to 70 nm thick using an ultramicrotome and stained with uranyl acetate and lead citrate (Wang et al., 2012). The tissues of leaves were placed on Cu-based grids and examined by TEM (JEM-1400 PLUS, Japan). Upon harvest, sunflowers were divided into shoots and roots. The roots from all treatments were thoroughly rinsed with tap water, and the weighed subsamples of fresh roots were used for mycorrhizal colonization assessment by the grid-line intersect method (Giovannetti and Mosse, 1980) after clearing with 10% KOH and staining with trypan blue. Shoots and roots were weighed after oven-drying at 70 °C for 48 h. Subsamples of dried and ground shoots and roots (1 and 0.5 g with the L and H soils, respectively) were digested by conc. nitric acid, followed by ICP-OES to measure tissue Cd, Cu, Pb, Cr, Zn, Ni and P concentrations (Hanson, 1950). Both blank and standard reference material [GBW10049 (GSB27), IGGE] were included for quality assurance. The recovery rates of HMs in tissues were between 64.0% and 130.3%. To this end, the P acquisition (the total amount of P in sunflowers per pot) and uptake of HMs (the total amount of each HM in sunflower shoots per pot) were calculated. The AM fungal inoculation responsiveness (MIR) is expressed as the percentage increase in plant biomass, and P concentration and acquisition, and was calculated using the following equation: MIR = [D(+M) − D(−M)] / D(−M) × 100%, where D(+M) and D (−M) are data from plants with and without AM fungal inoculation, respectively.

2.5. Soil chemical and biochemical property analysis

Table 1 The pH, and the total and DTPA-extractable concentrations of heavy metals (HMs) of the tested soils. Soil

Lightly HM-enriched soil (L)

Heavily HM-contaminated soil (H)

a b

pH

HM

5.52 Cd Cu Pb Cr Zn Ni 8.06 Cd Cu Pb Cr Zn Ni

Total concentration (mg kg−1)

Standard (mg kg−1)a

DTPA-extractable concentration (mg kg−1)b

0.389 61.0 53.6 108 163 44.4 33.7 3444 2883 386 14,437 373

0.3 50 250 150 200 40 0.6 100 350 250 300 60

0.118 13.3 7.01 – 15.4 3.65 0.139 53.7 3.16 – 86.4 0.175

pH b 6.5 for the L soil, pH N 7.5 for the H soil (SEPAC, 1995); Cr cannot be detected by the method.

Soil samples were air-dried and homogenized by sieving through a 2 mm mesh sieve. Soil pH, phosphatase activity, available P concentration, total Cr concentration, and total and DTPA-extractable concentrations of Cd, Cu, Pb, Zn, and Ni, were tested according to those methods introduced in the paragraph for soil preparation. Soil phosphatase activity was determined by incubation at 37 °C with citrate buffer (pH 7) according to the method of Tabatabai (1982), and is given in units of mg pnitrophenol produced g−1 soil 24 h−1. All these results were expressed on an oven-dried soil weight basis by correcting for water content in the soil (105 °C, 7 h).

2.6. Statistical analysis Data means and standard deviations were computed with Excel 2013. The analysis of variance (ANOVA) was based on Duncan's new multiple range method. Independent-Samples t-tests were applied to compare the results between the L soil and the H soil. Statistical significance was determined at the 95% level (P b 0.05). Both ANOVA and ttest were computed with SPSS19.0.


3. Results 3.1. Mycorrhizal colonization, plant biomass, and P concentration and acquisition of sunflower Mycorrhization in sunflower roots was shown in all treatments, even in the controls without AM fungal inoculation (Fig. 1a). The mycorrhizal colonization rate, and the biomasses (Fig. 1b) and P concentrations (Fig. 1c) of both shoots and roots, as well as the P acquisitions (Fig. 1d) of sunflower grown in the H soil, were all significantly smaller (P b 0.05) than those in the L soil. Both Fm and Fc inoculation significantly increased (P b 0.05) the root mycorrhizal colonization rates of sunflower grown either in the H or in the L soils, but only significantly elevated (P b 0.05) the biomasses and P concentrations of both shoots and roots, and the P acquisitions of sunflower grown in the H soil. Specifically, the MIRs of shoot biomass, root biomass, shoot P concentration, root P concentration, and P acquisition of sunflower grown in the H soil by Fm and Fc were 157–184%, 152–183%, 12–18%, 22–29%, and 214%, respectively. In addition, there were no significant differences between the Fm- and Fc- inoculated treatments with both H and L soils. 3.2. The tissue concentration and shoot uptake of heavy metals by sunflower Without AM fungal inoculation, the Cd, Cu, Pb, Cr, Zn, and Ni concentrations of sunflower grown in the H soil were significantly larger (P b 0.05) than those in the L soil, except for root Cr and shoot Ni (Fig. 2). For the L soil, there were no significant differences in HM concentrations either in shoots or in roots among the control and AM-inoculated

Fm

285

treatments. For the H soil, compared with the control, both Fm and Fc inoculations significantly decreased (P b 0.05) the concentrations of Cd, Cr, Zn and Ni in sunflower shoots. Fc and Fm also significantly reduced (P b 0.05) shoot Cu and Pb concentration, respectively. However, AM fungal inoculations had no significant effects on root HM concentrations of sunflower grown in the H soil. In addition, there were no significant changes in the shoot uptake of HMs by sunflower grown in the L soil in response to either Fm or Fc inoculation (Fig. 3). For the H soil, both Fm and Fc inoculations significantly increased (P b 0.05) the shoot uptake of Cd, Cu, Pb, Zn, and Ni, while Fm inoculation also significantly decreased (P b 0.05) the shoot uptake of Cr. 3.3. The CAT activity and TEM images of plant leaves Without AM fungal inoculation, there were no significant differences in the CAT activities of sunflower leaves between the L and H soils (Fig. 4a), but the thylakoid layered structure in the leaves of sunflower grown in the H soil were impaired and not visible (Fig. 4b, c). For the H soil, AM fungal inoculation significantly increased (P b 0.05) the CAT activity (Fig. 4a) and alleviated the toxicity symptoms of HMs in the thylakoid structure of leaves (Fig. 4d). 3.4. Soil pH, phosphatase activity, total and available P, and total and DTPAextractable heavy metals For the L soil, Fm rather than Fc inoculation significantly decreased (P b 0.05) soil pH, and significantly increased (P b 0.05) soil available P and DTPA-extractable Zn concentrations, but had no significant effects on soil phosphatase activity (Table 2) and the total and DTPA-extractable

Fc

Fig. 1. Mycorrhizal colonization (a), plant biomass (b), P concentration (c), and total P acquisition per pot (d). L, lightly heavy metal (HM)-enriched soil; H, heavily HM-contaminated soil; Control, non-mycorrhizal inoculum; Fm, inoculation with Funneliformis mosseae; Fc, inoculation with F. caledonium. Vertical T bars indicate standard deviations. Bars not topped by the same letter within each soil indicate a significant difference in values (P b 0.05). *, ** or *** indicate P b 0.05, P b 0.01 or P b 0.001 for the same treatments between L and H soils.


a

A A A X X ** X * * H

15

L

-1 )

Plant Cr concentration (mg kg Root Shoot

50 40 30 20

y

a a

a

y

0

20 40 60 80

30 20

A A

100

L

A

a

a

10 0 25

L

1500

*** x

1000

a

H

X

** *** y y

a

0 200 400

A

A A

4000

X X

X X * X * * H

3000

a

8000

L

20

*** x *** *** y xy

-1 ) 2 1 0

20

A A A

500

c

10

a

2000

e

** x

*** x *** xy *** y

40

50 1000

20

d

50

Plant Pb concentration (mg kg Shoot Root

a

*** *** y y

b

X X X ** * * H

a

a

a

A A A

400 X *

600 L

f

10

)

1.0 0.5 0.0 0.5 1.0 1.5 10

a

Fc

8 6

-1

5

Fm *** x

Plant Ni concentration (mg kg Root Shoot

Control

-1 Plant Cu concentration (mg kg ) Root Shoot

10

Plant Zn concentration (mg kg -1) Root Shoot

15

Plant Cd concentration (mg kg Root Shoot

a -1 )

286

a a

H

a

4

** x y

2

0 20 40 60 80 100 120

X X * **

A

*** y

A A

L

X X X * * * H

Fig. 2. Plant Cd (a), Cu (b), Pb (c), Cr (d), Zn (e), and Ni (f) concentrations. L, lightly heavy metal (HM)-enriched soil; H, heavily HM-contaminated soil; Control, non-mycorrhizal inoculum; Fm, inoculation with Funneliformis mosseae; Fc, inoculation with F. caledonium. Vertical T bars indicate standard deviations. Bars not topped by the same letter within each soil indicate a significant difference in values (P b 0.05). *, ** or *** indicate P b 0.05, P b 0.01 or P b 0.001 for the same treatments between L and H soils.

concentrations of most HMs (Table 3). For the H soil, AM fungal inoculations did not change soil pH, and the concentrations of soil available P, total Cu, total Cr, and DTPA-extractable Cu. However, both Fm and Fc inoculations significantly decreased (P b 0.05) soil total Ni concentrations. In addition, Fc rather than Fm significantly increased (P b 0.05) soil DTPA-extractable Pb, Zn, and Ni concentrations and decreased soil total Cd, Pb, and Zn concentrations. Meanwhile, Fc inoculation significantly increased (P b 0.05) soil phosphatase activity, while Fm inoculation only significantly decreased (P b 0.05) soil DTPA-extractable Cd concentration. 4. Discussion 4.1. Changes in soil quality and plant growth in response to WEEE recycling In the present study, the RD site, rather than the NF site, had been heavily contaminated by HMs due to the dumping of WEEE-recycling residues, and the concentrations of HMs in the soil varied from 1.5 to 56 times larger than the national standard (SEPAC, 1995). Leung et al. (2006) also found that the dumping of WEEE-recycling residues, such as printer roller, could lead to soil HM contamination. Thus, the deposition of WEEE-recycling residues onto the soil surface is a more direct and strong way to contaminate soil (Damrongsiri et al., 2016). Consequently, the RD site was no longer suitable for agricultural production, and farming activities decreased. Therefore, the application of P fertilizer was reduced or even stopped, and the concentrations of the total and the available P in the RD site (i.e., H soil) were greatly smaller than those in the NF site (i.e., L soil). In addition, the pH and the organic C concentration in the H soil were much higher than those in the L soil. The reasons for these changes are currently unclear and require further studies. WEEE-recycling residues usually contain a large concentration

of organic pollutants, which may lead to increased concentrations of organic matter in the soil (Leung et al., 2006), and large concentrations of HMs and organic pollutants in the soil could generate inhibitory effects on the activities of microbes related to C turnover via decreasing organic C mineralization (Dai et al., 2004), contributing to a lower soil organic C loss. The more organic matter in the soil can sorb and retain more HMs (Covelo et al., 2007), and the higher soil pH can reduce the solubility and mobility of HMs (Ho et al., 2012; Wu et al., 2015). Thus, the ratios of DTPA-extractable to the total concentration of HMs in the H soil were 14 to 175 times smaller than in the L soil, and the largest difference between soils was contributed by Ni. The large concentrations of HMs in the H soil led to increases in HM concentrations in sunflower shoots and roots (Fig. 2). The H soil, but not the L soil, induced the obvious visual symptoms that the leaves became dry and black due to HM phytotoxicity. Visual symptoms have been observed only in substrates with large concentrations of HMs (de Andrade et al., 2008; Hassan et al., 2013). As a result, sunflower has a certain tolerance to HM stress. However, the thylakoid structure of chloroplasts was impaired (Fig. 4c). Thylakoids are distributed in the chloroplast stroma, and the damage can affect plant photosynthesis and biomass (Gill et al., 2015). Thus, the biomasses of plants were reduced (Fig. 1b). These results can reflect the toxicity of HMs on plants. However, the Ni concentrations of sunflower grown in the H soil were smaller than those in the L soil. The soil DTPA-extractable metals represent the bioavailable fraction of metals in soil. Even though the total concentration of Ni in the H soil was larger than that in the L soil, the DTPA-extractable Ni may decrease with the increasing soil pH and organic matter content. The total P concentrations in plant shoots and roots were reduced due to the small P concentration in the H soil. Moreover, the soils tested in this experiment were not sterilized, and mycorrhization could be found in the sunflower without AM fungal


Fc

a

a * ** x y

20 *** z

10

0

b

600

c

a a

a

400

200

*** *** x x *** y

0 L

H

80 a

-1

a

30

Fm

Plant Pb uptake (µg pot )

Control

Plant Cu uptake (µg pot-1)

40

-1

Plant Cd uptake (µg pot )

a

287

a

60 a

x

40

y *** z

20

0

L

H

L

H

Fig. 3. Plant Cd (a), Cu (b), Pb (c), Cr (d), Zn (e), and Ni (f) uptake. L, lightly heavy metal (HM)-enriched soil; H, heavily HM-contaminated soil; Control, non-mycorrhizal inoculum; Fm, inoculation with Funneliformis mosseae; Fc, inoculation with F. caledonium. Vertical T bars indicate standard deviations. Bars not topped by the same letter within each soil indicate a significant difference in values (P b 0.05). *, ** or *** indicate P b 0.05, P b 0.01 or P b 0.001 for the same treatments between L and H soils.

inoculation, but the colonization rates were extremely low in the H soil (Fig. 1a). This indicated that large concentrations of HMs in the soil significantly inhibited the colonization of indigenous AM fungi. However, both the promotion (Jarrah et al., 2014) and no effect (Hassan et al., 2013; Rozpadek et al., 2014; Arias et al., 2015) of HMs on AM fungal colonization have also been reported. This may be the result of “functional diversity” with different symbioses of AM fungal strains and plant species (Liu et al., 2015). 4.2. Changes in plant growth and HM-uptake in response to mycorrhizal inoculation A number of works so far have focused on the influences of AM fungal inoculations on HM absorption by various plants, most of which were based on the conditions of soil sterilization (Andrade et al., 2010; Chan et al., 2013; Zhan et al., 2016). More importantly, there was a lack of AM fungal research on heavily contaminated soils with various HMs caused by WEEE recycling. In this study, both Fm and Fc could form a good symbiotic relationship with sunflower in both L and H soils (Fig. 1a). HMs had no inhibitory effects on exogenous AM fungi, and the reason may be that the AM fungal inocula were inoculated as a layer, but not completely mixed with the soil. Nevertheless, a large concentration of soil P may have an adverse effect on mycorrhizal colonization in host roots (Wang et al., 2017), and plants are more dependent on AM fungi in P-poor soil than in P-fertilized soil (Hu et al., 2009). Thus, the colonization of Fm to sunflower in the H soil was significantly higher than that in the L soil, and Fc had a similar trend (Fig. 1a). The root with AM fungal inoculation often enhances P absorption and plant growth by tapping a larger volume of soil than that without inoculation for the relatively immobile P or solubilizing normally insoluble P sources through the excretion of various organic acids (Hu et al., 2016). Meanwhile, the higher P absorption by plants

upon AM fungal inoculations also seemed to be due to the elevation of soil phosphatase activity (notably Fc) (Table 2), which may involve AM fungi directly and indirectly: AM propagules can synthesize such enzymes, and mycorrhizal roots may release more root exudates containing enzymes due to the improved nutrition and/or larger root system (Wang et al., 2006; Hu et al., 2014). As a result, the observed large increases in P acquisition (Fig. 1d) and thus plant biomass (Fig. 1b) of sunflower following both Fm and Fc inoculations were mostly due to the enhanced root mycorrhizal colonization rate (Fig. 1a) and soil phosphatase activity (notably Fc) (Table 2). For the H soil, AM fungal inoculations substantially reduced the concentrations of HMs in the shoots, but not in the roots, of sunflower relative to the non-inoculated treatment (Fig. 2). This indicated that the translocation efficiencies of HMs from root to shoot were decreased. However, Chen et al. (2007) found that AM fungal colonization significantly decreased As concentrations in both shoots and roots, due to the ‘dilution effect’ by improving P nutrition and plant biomass. Thus, the dilution effect may be another cause of the decreased HM concentrations in the shoots in this study. Additionally, the increase in the peroxidase level is thought to protect plant cells from free radical oxidation, allowing the plant to adapt to the stressor (Latef, 2013), and AM fungi can activate the expression of genes encoding some components of its antioxidant network to address this imbalanced redox status and repair the damage (Ferrol et al., 2016). This suggests that AM fungi can reduce HM stress to host plants by increasing the activity of antioxidant enzymes (Rozpadek et al., 2014). In this study, AM fungal inoculation increased the CAT activity of the leaves of sunflower grown in the H soil, which was similar to Fm-inoculated pigeon pea (Cajanus cajan (L.) Millsp) in Cd- and Pb- contaminated soils (Garg and Aggarwal, 2011), and F. constrictum-inoculated marigold (Tagetes erecta L.) in Cd-added soils (Liu et al., 2011). The outcomes of TEM were consistent with the increased activity of CAT in this study. The TEM images showed that the

288


Fig. 4. The catalase (CAT) activities (a) and transmission electron microscope (TEM) images of chloroplasts (b, c, and d) in the leaves of sunflower. LControl, sunflower grown in the lightly heavy metal (HM)-enriched soil (L) with non-mycorrhizal inoculum; HControl, sunflower grown in the heavily HM-contaminated soil (H) with non-mycorrhizal inoculum; HM, sunflower grown in the H soil with AM fungal inoculum (M). Vertical T bars indicate standard deviations. Bars not topped by the same letter indicate a significant difference in values (P b 0.05).

structure of thylakoid in plant leaves were impaired by large accumulations of HMs (Fig. 4c); however, AM fungal inoculations alleviated the toxicity symptoms of HMs in thylakoid structure to a certain degree (Fig. 4d). AM fungi can alter soil composition and properties during metabolic processes, thereby altering the bioavailability of HMs (Leung et al., 2013). In this study, the soil DTPA-extractable concentrations of Pb, Zn, and Ni (especially by Fc), and the HM uptake of sunflower grown in the H soil were increased by AM fungal inoculation. The increased HM uptake indicated that sunflower could take more HMs from the soil, so the concentrations of HMs in the soils tended to decrease (Table 3). Therefore, the combination of AM fungi and sunflower had the potential for phytoextraction of HMs from WEEE-recycling sites. Although it was shown that AM fungi can decrease the HM concentrations in shoots and alleviate the stress of HMs to some extent, visual symptoms were still observed in the leaves of sunflowers even with AM fungal inoculations. To achieve a better understanding and effect of the enhancement of AM fungi on the reduction of HM stress to plants,

further studies on basic mechanisms and using field experiments are essential. Additionally, sufficient AM inocula are too difficult to obtain for inoculation in the field, thus the preparation and application of mycorrhizal seedlings seems to be more vital in future studies. 5. Conclusions Intensive WEEE recycling, notably residue dumping, resulted in heavy contamination with varied HMs in the soil, which led to the accumulation of HMs in plants, damaging organelles and inhibiting plant growth. AM fungal inoculations elevated P acquisition and promoted plant growth. Meanwhile, inoculations of AM fungi can decrease the shoot HM concentrations, increase CAT activity, and alleviate the toxicity symptoms of HMs in the thylakoid structure of leaves. Additionally, AM fungal inoculations significantly elevated the HM uptake of sunflower and tended to decrease the total concentrations of HMs in the soil. However, only Fc provided a significant elevation in soil phosphatase activity, and Fc rather than Fm significantly increased the DTPA-

Table 2 Soil pH, phosphatase activity, and available P concentration with or without AM fungal inoculation. Soil

Treatment

pH

Phosphatase activity (mg p-nitrophenol g−1 24 h−1)

Available P concentration (mg kg−1)

L

Control Fm Fc Control Fm Fc

5.72 ± 0.04a 5.63 ± 0.02b 5.68 ± 0.02ab 8.37 ± 0.02x*** 8.38 ± 0.01x*** 8.36 ± 0.03x***

3.23 ± 0.12a 3.21 ± 0.23a 3.06 ± 0.21a 0.11 ± 0.01y*** 0.12 ± 0.01y*** 0.15 ± 0.01x***

80.6 ± 1.57b 83.5 ± 1.69a 81.2 ± 1.74ab 1.95 ± 0.18x*** 2.01 ± 0.34x*** 2.56 ± 0.52x***

H

L, lightly heavy metal (HM)-enriched soil; H, heavily HM-contaminated soil; Control, non-colonization; Fm, inoculation with Funneliformis mosseae; Fc, inoculation with F. caledonium. Values in the table are means of four replications ± standard deviations. Values within the same column for each soil not followed by the same letter indicate a significant difference (P b 0.05). *, ** or *** indicate P b 0.05, P b 0.01 or P b 0.001 for the same treatments between L and H soils.


289

Table 3 The total and DTPA-extractable concentrations of heavy metals in soils with or without AM fungal inoculation. Soil

Total concentration

L

H

DTPA-extractable concentration

L

H

Treatment

Control Fm Fc Control Fm Fc Control Fm Fc Control Fm Fc

Cd

Cu

Pb

Cr

Zn

Ni

(mg kg−1)

(mg kg−1)

(mg kg−1)

(mg kg−1)

(mg kg−1)

(mg kg−1)

0.36 ± 0.03a 0.37 ± 0.02a 0.36 ± 0.02a 31.0 ± 0.35x*** 28.5 ± 2.50xy*** 27.7 ± 1.02y*** 0.12 ± 0.01a 0.12 ± 0.01a 0.13 ± 0.001a 0.15 ± 0.003x* 0.13 ± 0.009y 0.15 ± 0.007x*

57.9 ± 0.84a 61.0 ± 4.21a 60.2 ± 2.14a 3546 ± 528x** 3070 ± 338x*** 2871 ± 416x** 12.1 ± 0.9a 12.4 ± 0.49a 12.0 ± 0.60a 54.6 ± 3.81x*** 53.3 ± 1.00x*** 56.0 ± 3.73x***

52.4 ± 1.12a 54.4 ± 2.25a 54.7 ± 5.41a 2912 ± 228x** 2732 ± 388xy** 2295 ± 249y*** 4.49 ± 1.54a 4.44 ± 1.15a 3.51 ± 1.53a 3.46 ± 0.31y 3.60 ± 0.06xy 4.05 ± 0.49x

100 ± 0.46a 101 ± 0.63a 103 ± 6.30a 361 ± 41.8x*** 307 ± 9.80x*** 321 ± 53.7x*** – – – – – –

161 ± 2.45a 165 ± 5.63a 163 ± 5.69a 13,440 ± 147x*** 12,312 ± 972xy*** 10,935 ± 898y*** 13.58 ± 0.31b 14.76 ± 0.38a 13.94 ± 0.71b 85.2 ± 6.02y*** 88.4 ± 3.07xy*** 99.1 ± 11.86x**

43.1 ± 0.16a 44.2 ± 0.22a 44.1 ± 2.15a 305 ± 15.9x** 264 ± 10.8y*** 264 ± 30.3y** 3.28 ± 0.11a 3.44 ± 0.12a 3.32 ± 0.16a 0.20 ± 0.01y*** 0.21 ± 0.02xy*** 0.23 ± 0.01x***

L, lightly heavy metal (HM)-enriched soil; H, heavily HM-contaminated soil; Control, non-colonization; Fm, inoculation with Funneliformis mosseae; Fc, inoculation with F. caledonium. Values in the table are means of four replications ± standard deviations. Values within the same column for each soil not followed by the same letter indicate a significant difference (P b 0.05). *, ** or *** indicate P b 0.05, P b 0.01 or P b 0.001 for the same treatments between L and H soils.

extractable HMs. These results suggested the potential application of sunflower associated with AM fungi (notably Fc) for reducing HM stress and promoting phytoextraction of HMs from heavily contaminated field soil caused by WEEE recycling.

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