Mycorrhizal Inoculation Affects Pb and Cd Accumulation and Translocation in Pakchoi (Brassica chinensis L.)

Pedosphere 26(1): 13–26, 2016 doi:10.1016/S1002-0160(15)60018-2 ISSN 1002-0160/CN 32-1315/P c 2016 Soil Science Society of China ⃝ Published by Elsevier B.V. and Science Press

Mycorrhizal Inoculation Affects Pb and Cd Accumulation and Translocation in Pakchoi (Brassica chinensis L.) WU Zhipeng1,2 , WU Weidong2 , ZHOU Shenglu1,∗ and WU Shaohua1 1 School

of Geographic and Oceanographic Sciences, Nanjing University, Nanjing 210046 (China) of Education Key Laboratory of Protection and Development Utilization of Tropical Crop Germplasm Resources, Hainan University, Haikou 570228 (China) 2 Ministry

(Received June 24, 2015; revised November 2, 2015)

ABSTRACT Heavy metal (HM) contamination in soils is an environmental issue worldwide that threatens the quality and safety of crops and human health. A greenhouse experiment was carried out to investigate the growth, mycorrhizal colonization, and Pb and Cd accumulation of pakchoi (Brassica chinensis L. cv. Suzhou) in response to inoculation with three arbuscular mycorrhizal (AM) fungi (AMF), Funneliformis mosseae, Glomus versiforme, and Rhizophagus intraradices, aimed at exploring how AMF inoculation affected safe crop production by altering plant-soil interaction. The symbiotic relationship was well established between pakchoi and three AMF inocula even under Pb or Cd stress, where the colonization rates in the roots ranged from 24.5% to 38.5%. Compared with the non-inoculated plants, the shoot biomass of the inoculated plants increased by 8.7%–22.1% and 9.2%–24.3% in Pb and Cd addition treatments, respectively. Both glomalin-related soil protein (GRSP) and polyphosphate concentrations reduced as Pb or Cd concentration increased. Arbuscular mycorrhizal fungi inoculation significantly enhanced total absorbed Pb and Cd (except for a few samples) and increased the distribution ratio (root/shoot) in pakchoi at each Pb or Cd addition level. However, the three inocula significantly decreased Pb concentration in pakchoi shoots by 20.6%–67.5% in Pb addition treatments, and significantly reduced Cd concentration in the shoots of pakchoi in the Cd addition treatments (14.3%–54.1%), compared to the non-inoculated plants. Concentrations of Pb and Cd in the shoots of inoculated pakchois were all below the allowable limits of Chinese Food Safety Standard. The translocation factor of Pb or Cd increased significantly with increasing Pb or Cd addition levels, while there was no significant difference among the three AMF inocula at each metal addition level. Meanwhile, compared with the non-inoculated plants, AMF inocula significantly increased soil pH, electrical conductivity, and Pb or Cd concentrations in soil organic matter in the soils at the highest Pb or Cd dose after harvest of pakchoi, whereas the proportion of bioavailable Pb or Cd fraction declined in the AMF inoculated soil. Our study provided the first evidence that AM fungi colonized the roots of pakchoi and indicated the potential application of AMF in the safe production of vegetables in Pb or Cd contaminated soils. Key Words:

arbuscular mycorrhizal fungi, bioavailable Cd and Pb, colonization, heavy metal, phytoavailability

Citation: Wu Z P, Wu W D, Zhou S L, Wu S H. 2016. Mycorrhizal inoculation affects Pb and Cd accumulation and translocation in Pakchoi (Brassica chinensis L.). Pedosphere. 26(1): 13–26.

INTRODUCTION Although Pb and Cd are not an essential element for plants, they get easily absorbed and accumulated by crops. High concentrations of Pb and Cd can not only make detrimental effects on soil biological activities and plant metabolism (Kabata-Pendias, 2010; Haouari et al., 2012), but also pose a significant risk to human health via the soil-crop-human exposure pathway. Lead and Cd are mainly originated from mining, electroplating, pigments, incinerators after product disposal, and metallurgical industries (J¨arup, 2003; Sharma and Dubey, 2005). Vegetables are important component of human fo∗ Corresponding

author. E-mail: [email protected].

odstuffs around the world, whose consumption is one of the most prominent pathways of heavy metals entering the food chain (Wang et al., 2004). Brassicaceae family vegetables are considered as a critical food crop source of the human dietary system in China, Japan, India, and European Union (Cartea et al., 2010). Pakchoi (Brassica chinensis L. cv. Suzhou) is a widely cultivated leafy vegetable in China, which is one of the Brassica species that show an ability to absorb and accumulate high concentrations of heavy metals (Wu et al., 2013). Thus, the safety and quality of pakchoi has had an increased concern in recent years. However, heavy metal accumulation in plant tissues is influenced by its bioavailability in soils, which is associated not

Z. P. WU et al.

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only with soil physicochemical conditions (Akkajit and Tongcumpou, 2010), but also with microbial activities (Medina et al., 2005). Arbuscular mycorrhizal fungi (AMF), occurring in the soil of almost all ecosystems including contaminated soils, are commonly associated with a majority of terrestrial plants by forming the mutual symbiosis (Smith and Read, 2010). Mycorrhizal mycelium around the root acts as an intermediary between the soil and the plant, absorbing nutrients and water from soil and delivering them to the host root (Reinhardt, 2007). In turn, mycelium can obtain P from the plant in order to complete their metabolism. Arbuscular mycorrhizal fungi can protect the plant growing in metal-contaminated soils against environmental stress by immobilizing metal ions to hyphal cell walls (Turnau et al., 1994) and polyphosphate particles in the cell lumen (Turnau et al., 1993), sequestering metal ions in the rhizospheric soils via chelation with glomalin (Gonz´alez-Ch´ avez et al., 2004) or alteration of soil conditions (Li and Christie, 2001), and increasing shoot biomass through improving essential nutritional status (e.g., P) (Rivera-Becerril et al., 2002) to alleviate metal stress. Most studies reported that AMF frequently reduced plant uptake and/or the phytotoxic effects of soil heavy metals, although in some cases enhanced aerial uptake of toxic metals may be observed in the hyperaccumulator (Vogel-Mikuˇs et al., 2006). To date, AMF have been inoculated to plants for agricultural food and medicine safety, such as Glomus caledonium (Nicol. & Gerd.) Trappe & Gerdemann for maize (Zea mays L.) growing on Cu contaminated soil (Shen et al., 2004), G. caledonium (Nicol. & Gerd.) Trappe & Gerdemann and G. versiforme (Daniels & Trappe) for bashfulgrass (Mimosa pudica L.) growing on Cd-contaminated soil (Hu et al., 2014). Recent advances in AMF application for many crop production and safety have been investigated. Compared with the other crops, Brassicaceae family plants are hard to establish a symbiotic relationship with AMF under the natural conditions (Smith and Read, 2010). Only a few literatures have documented that some Brassica plants can be successfully colonized by added AMF such as cauliflower (Brassica oleracea L. var. italica Plenck) (Purakayastha et al., 1998), cabbage (Brassica oleracea var. capitata) (Nelson and Achar, 2001) and rape (Brassica napus L.) (Li, 2007). However, less information is reported on AMF infecting pakchoi which is a widely cultivated leafy vegetable in China and its application in the context of vegetable production from metal-contaminated

soils. Therefore, we hypothesized that AMF could colonize and increase pakchoi growth but decrease its Pb and Cd uptake, and their influence could be different from fungal species. A pot experiment was carried out by inoculating three AMF inocula, including Funneliformis mosseae, G. versiforme, and Rhizophagus intraradices, to pakchoi to investigate: i) the performance of three AMF inocula in soils under different levels of Pb or Cd addition; ii) mycorrhizal influence on pakchoi growth and Pb and Cd accumulation and translocation in pakchoi; and iii) the main soil factors influencing these parameters. MATERIALS AND METHODS Soil preparation The topsoil sample (0–20 cm) was collected and mixed from uncontaminated garden land at a suburb of Haikou, Hainan Province, China. This site is characterized by a tropical marine monsoon climate, with a mean annual temperature of 23.8 ◦ C and an average annual precipitation of 1 664 mm. The loam soil at the experimental site is classified as Udept derived from basalt. Prior to the experiment, soil samples were air-dried, ground with an agate pestle, homogenized, sieved through a 5-mm sieve, and then sterilized in an autoclave at 121 ◦ C for 1 h on two successive days. Selected soil properties were determined with three subsamples. Soil pH and electrical conductivity (EC) were measured in 1:2.5 and 1:5 soil/water suspensions (weight/volume), respectively. Soil organic matter (SOM) was measured using the K2 Cr2 O7 -heating method as described by Sarkar and Haldar (2005). Soil clay contents (< 0.002 mm, hydrometer method), cation exchange capacity (CEC, 1 mol L−1 ammonium acetate, pH 7.0), and free Fe/Mn/Al oxide (Dithionite/citrate/bicarbonicum extractant) were analyzed according to Lu (1999). Background Pb and Cd concentrations in soil were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Fisher-X series, Thermo Fisher Scientific Inc., USA). The results are described in Table I. Both background Pb and Cd concentrations were far below the 2nd level criterion of environmental quality standard for agricultural soils with pH < 6.5 in China (250 mg kg−1 for Pb and 0.3 mg kg−1 for Cd) (GB15618-1995). Mycorrhizal inoculum Three AMF inocula, F. mosseae, G. versiforme, and R. intraradices (Sch¨ ußler and Walker, 2011), were

MYCORRHIZAL INOCULATION AFFECTS METALS IN PAKCHOI

15

TABLE I Selected propertiesa) of the soil used for the pot experiment pH

EC cm−1

6.45 a) EC

µS 66.4

Clay kg−1

g 164

SOM g−1

mg 13.8

CEC cmol 4.86

Fe oxide kg−1 30.2

Al oxide mg 43.7

Mn oxide

Background Cd

g−1 0.45

0.06

Background Pb

mg kg−1 18.8

= electrical conductivity; SOM = soil organic matter; CEC = cation exchange capacity.

used in the experiment. The propagules of the three inocula were collected from the sugarcane field in Danzhou, Hainan Province, China. Then they were preserved at the Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences in Danzhou. Arbuscular mycorrhizal fungi inocula were propagated on stylosanthes (Stylosanthes guianensis Sw.) grown in an autoclaved substrate (121 ◦ C for 1 h on three successive days) for 3 months. All inocula were air-dried and passed through a 2-mm sieve before inoculation. Experimental layout Soil samples (2 kg) were placed in each plastic pot (22 cm diameter × 20 cm height) after mixing thoroughly with Pb(NO3 )2 or CdSO4 ·5H2 O solutions to achieve two levels of Pb (Pb1 = Pb addition at 125 mg kg−1 and Pb2 = Pb addition at 250 mg kg−1 ) and Cd (Cd1 = Cd addition at 0.9 mg kg−1 and Cd2 = Cd addition at 1.5 mg kg−1 ), respectively. Soil without metal addition was used as a control. The soil was then left interiorly in a cool place to equilibrate for about one month in order to allow natural equilibration of the various sorption in the soil. During this timeframe, soil water was maintained at 80% of the maximum water holding capacity. All treatments were arranged in a randomized complete block design with twelve replicates. Seeds of pakchoi (Brassica chinensis L. cv. Suzhou) were sterilized with 0.5% NaClO and rinsed twice with ultrapure water. Afterwards, the seeds (0.5 g) mixed with AMF inocula (5 g) were sown directly to the soil in each pot. In total, there were four treatments for each metal (Pb or Cd) addition treatment, including the non-inoculated control (−M) and three inoculated treatments (+M), i.e., inoculation with F. mosseae, G. versiforme, and R. intraradices, with three replicates. The procedural controls of no plants in pots were also designed. The seedlings were cultivated for 10 weeks in a controlled environment with a photoperiod of a 16 h/8 h (light/dark), a temperature of 25 ◦ C/20 ◦ C (light/dark), and 75% relative humidity. Seedlings were thinned to three per pot after two weeks. The

plants were watered every 2 d to maintain moderate soil moisture until the end of the experiment. Plant samples (roots and shoots) were also taken from different pots after the harvest for the analysis of Pb, Cd and AMF. Soil samples (about 500 g) were also collected from each pot after the harvest for determination. Mycorrhizal colonization and polyphosphate analysis After the harvest, the pakchoi roots with diameter ≤ 1 mm were carefully washed several times with deionized water, chopped into 1-cm long pieces, and then fixed in acetic acid/formalin/ethanol (AFE) solution for 24 h and stored at 4 ◦ C. After AFE fixation, pakchoi roots were stained with 0.l g L−1 acid fuchsin according to Phillips and Hayman (1970). Mycorrhizal colonization (MC, %) was evaluated using the grid-line intersect method (McGonigle et al., 1990): MC = (Np /No ) × 100

(1)

where Np and No are the number of mycorrhizal root pieces and total number of observed root pieces, respectively. Polyphosphate concentration in hyphae was assessed according to Ezawa et al. (2001) with the minute modifications. Extraradical hyphae were separated from the root, stained with 0.05% (weight/volume) KCl/HCl buffer (TBO, 50 mmol L−1 , pH = 1.0) for 20 min, rinsed in TBO, and spread on a glass slide. Then, percentage of metachromatic hyphal length was measured under a compound light microscope based on the grid line intersect method. Soil heavy metal analysis Soil samples (0.1 g, < 0.149 mm) were digested with an acid mixture (HNO3 /H2 O2 /HF, 3/2/1, volume/volume/volume) in a microwave digestion system (SINED, MDS-6) for the analysis of the total Pb and Cd. Bioavailable Cd and Pb in the soil were extracted with a mixed extractant containing 50 mmol L−1 diethylene triamine penta-acetic acid (DTPA), 10 mmol L−1 acid-triethanolamine (TEA), and 10 mmol L−1 CaCl2 (pH = 7.3) according to the method of Wu

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et al. (2014). Soil was mixed with the extractant (1:5, weight/volume) in plastic tube, shaken for 2 h, centrifuged at 2 500 × g, and then filtered into a plastic jar for further analyses. Metals in soil SOM (SOM-metals) were extracted according to Tessier et al. (1979). Total, bioavailable, and SOM-Pb and Cd concentrations were analyzed by ICP-MS.

+M represent the non-inoculated control and treatments inoculated with AMF inocula, respectively. Mycorrhizal contribution rate (MCR) was used to evaluate the ability of AMF to influence Pb and Cd accumulation in plants:

Glomalin-related soil protein and bound to metal analysis

where A is the total amount of Pb or Cd in the shoots and roots (µg). The translocation factor (TF) was calculated to evaluate the influence of AMF on plant’s ability to translocate Pb and Cd from roots to shoots:

Glomalin is a stable and persistent glycoprotein produced in copious quantities by mycorrhizal fungi (Wu et al., 2014) and an alkaline-soluble glycoprotein that has been operationally quantified from diverse soils as glomalin-related soil protein (GRSP). In the study, GRSP was determined using the Bradford method with bovine serum albumin (BSA) as a standard according to Wright and Upadhyaya (1998) with minor modifications. Total GRSP (T-GRSP) in soil (0.5 g, < 0.25 mm) was extracted with 4 mL-sodium citrate buffer (0.02 mol L−1 , pH = 7.0) at 121 ◦ C for 30 min in an autoclave. The supernatant was separated by centrifugation at 8 000 × g for 20 min and filtrated into the volumetric flask. To determine GRSPbound Pb (GRSP-Pb) and Cd (GRSP-Cd), GRSP in the above supernatant was precipitated using 2 mol L−1 HCl up to pH 2.5, centrifuged at 8 000 × g for 20 min, redissolved in 0.5 mol L−1 NaOH, dialyzed against deionized water, and freeze-dried according to Gonz´alez-Ch´avez et al. (2004). Dried-GRSP was digested with 4 mL of concentrated HNO3 and 2 mL of concentrated H2 O2 and analyzed by ICP-MS. Plant analysis Fresh pakchois were firstly washed with deionized water, rinsed thoroughly with ultrapure water, and divided into roots and shoots. Fresh weight (FW) was recorded, and samples were dried at 105 ◦ C for 30 min and then 75 ◦ C in an oven for 48 h for determining the yield and metal concentrations. Fresh (3 g) and dry (0.3 g) roots or shoots were digested with concentrated acid mixture of HNO3 (2 mL) and H2 O2 (1 mL) in a microwave digestion system (MDS-6, Sineo, China). Concentrations of Pb and Cd were measured using ICP-MS. Mycorrhizal dependence (MD, %) was calculated to assess the ability of AMF to influence the growth of plants as follows: MD = (Y+M /Y−M − 1) × 100

(2)

where Y is the dry shoot biomass (g), while –M and

MCR = 100 × (A+M − A−M )/A+M

TF = Cshoot /Croot

(3)

(4)

where Cshoot and Croot represent the Pb or Cd concentrations in shoot (stem and leaves) and root (mg kg−1 ), respectively (Wu et al., 2013). Statistical analysis Data were analyzed with statistical package SPSS 19.0 and Origin 9.0. All data were described in the form of means ± standard deviations. Mycorrhizal development status (MC, MD, and polyphosphate and GRSP concentrations) and heavy metal (Pb and Cd) accumulation and translocation in pakchoi were analyzed by two-way analysis of variance (ANOVA) according to Duncan’s multiple-range tests (with a 95.0% confidence level) to assess the effects of AMF inoculation and heavy metal (Pb or Cd) addition levels. When the interaction of AMF inocula and heavy metal addition level were significant, comparisons among AMF inocula were performed using one-way ANOVA and Duncan’s test. Soil properties data at the end of experiment were also analyzed by one-way ANOVA according to Duncan’s multiple-range tests. RESULTS Mycorrhizal development status Arbuscular mycorrhizal fungi structures were successfully detected in and on the roots of inoculated pakchoi after 70 d (Fig. 1, Table II), while no AMF colonization was detected in the non-inoculated plant treatments. Mycorrhizal development status of F. mosseae, G. versiforme, and R. intraradices inocula in pakchoi are shown in Figs. 2 and 3. Overall, MC of all AMF inocula in Pb0 and Pb2 treatment were significantly higher than in Pb1 treatment (Fig. 2a). However, MD and GRSP and polyphosphate concentrations (except for G. versiforme inoculation) decreased with

MYCORRHIZAL INOCULATION AFFECTS METALS IN PAKCHOI

17

Fig. 1 Vesicles, intercellular hyphae, extraradical hyphae, arbuscules, and branched absorbing structures observed in and on the roots of pakchoi inoculated with Funneliformis mosseae after 70 d. TABLE II F value of two-way analysis of variance applied to mycorrhizal development status, using levels of metal (Pb and Cd) addition (LEpb and LECd ) and arbuscular mycorrhizal fungi (AMF) inocula (AMFI) as factors Mycorrhizal development status Mycorrhizal colonization Mycorrhizal dependence GRSPa) concentration Polyphosphate concentration

Pb addition treatment

Cd addition treatment

LEpb

AMFI

LEpb × AMFI

LECd

AMFI

LECd × AMFI

29.1*** 857.1*** 1 131.8*** 1 319.6***

6.8** 1 070.2*** 301.3*** 297.3***

7.9** 67.3*** 101.5*** 232.9***

650.2*** 983.9*** 995.1*** 1 417.5***

139.1*** 786.4*** 26.9*** 207.2***

101.8*** 74.3*** 46.6*** 47.6***

**, ***Significant at P < 0.01 and P < 0.001, respectively. soil protein.

a) Glomalin-related

increasing Pb level in soils (Fig. 2b, c, d). Polyphosphate concentration in pakchoi inoculated with G. versiforme was higher than those inoculated with F. mosseae and R. intraradices (Fig. 2d) in Pb0 and Pb1 treatments. Mycorrhizal dependence and GRSP concentration were higher in F. mosseae and R. intraradices inoculated pakchoi than in G. versiforme inoculated plants in all the Pb treatments. Mycorrhizal colonization (except for R. intraradices), MD, GRSP concentration (except for R. intraradices), and polyphosphate concentration were significantly reduced as Cd addition level in soils increased (Fig. 3). Among the three different AMF inocula, mycorrhizal colonization (except for the Cd0 treatment) and dependence of F. mosseae and R. intraradices were significantly higher than those of G. versiforme in all the Cd addition treatments (Fig. 3a, b). Mycorrhizal status differences of GRSP and polyphosphate concentrations were influenced by the Cd addition. Glomalin-related soil protein concen-

trations were significantly higher for R. intraradices than F. mosseae and G. versiforme in Cd0 and Cd2 treatments, while these were lower for R. intraradices than F. mosseae and G. versiforme in Cd1 treatment (Fig. 3c). Polyphosphate concentrations of F. mosseae and G. versiforme were significantly higher than those of R. intraradices in Cd addition treatments, whereas polyphosphate concentration of G. versiforme was highest in the control (Fig. 3d) Mycorrhizal contribution to Pb and Cd uptake and distribution in different organs Tables III and IV showed that both metal (Pb or Cd) addition level and AMF inocula significantly affected Pb and Cd uptake and distribution ratio of pakchoi plants. The total Pb or Cd concentration in both roots and shoots of both the non-inoculated and inoculated plants were increased significantly with the increasing Pb or Cd addition levels, respectively. The distribution ratio of Pb in both the non-inoculated and

18

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Fig. 2 Mycorrhizal colonization (a), mycorrhizal dependence (b), glomalin-related soil protein (GRSP) concentration (c), and polyphosphate concentration (d) of three arbuscular mycorrhizal fungi (Funneliformis mosseae, Glomus versiforme, and Rhizophagus intraradices) inocula in the rhizosphere of pakchoi after 70 d under different Pb addition treatments. Pb0 = no Pb addition; Pb1 = Pb addition at 125 mg kg−1 ; Pb2 = 250 mg kg−1 . Values are means with standard deviations shown by the vertical bars (n = 6). Bars with the same letter(s) indicate no significant differences at P < 0.05.

inoculated treatments was up to the largest value in Pb2 treatment (Table III), whereas the distribution ratio of Cd was raised significantly as Cd addition levels increased (Table IV). However, the effects of different mycorrhizal inocula on metal (Pb and Cd) uptake and distribution in plant seemed to be quite complex. On the whole, mycorrhizal inoculation could significantly enhance total metal (Pb and Cd) concentrations and distribution rates (root/shoot) in pakchoi compared to the non-inoculated treatments (Tables III and IV). When no Pb or Cd was added, MCR was the highest for G. versiforme, while it was significantly lower than that for F. mosseae and R. intraradices in the Pb (or Cd) addition treatments. The distribution ratio of Pb or Cd was higher for G. versiforme than for F. mosseae and R. intraradices in no Pb or Cd addition treatment. At any level of Pb or Cd addition, F. mosseae and R. intraradices were more helpful to facilitate redistribution of Pb from pakchoi roots to shoots than G. versiforme, while Cd distribution (root/shoot) was higher for plants inoculated with R. intraradices than plants inoculated with F. mosseae and G. versiforme.

Mycorrhizal influence on Pb and Cd accumulation and translocation in the roots and shoots We also investigated the effect of mycorrhizal inocula on the accumulation of Pb and Cd in pakchoi roots and shoots (Table V). Concentrations of Pb remained at 0.0368–1.3350 and 0.0291–0.2343 mg kg−1 FW in pakchoi roots and shoots after 70 d, respectively. Concentrations of Pb in roots were about 1.23–5.81 times higher than shoots. Concentrations of Pb were significantly lower in both roots and shoots of AMF inoculated pakchois in Pb addtion treatments when compared to non-inoculated pakchois. When no Pb was added, Pb concentrations in either the roots or shoots of noninoculated pakchois were significantly lower than those inoculated with F. mosseae. As for Cd, the range of Cd concentrations in roots was from 0.006 54 to 1.20121 mg kg−1 FW after 70 d (Table V), which equated to 1.24–6.10 times when compared with that of the corresponding shoots. In Cd addition treatment, the three AMF inocula significantly decreased Cd concentrations in roots and shoots of pakchoi, compared with noninoculated pakchois. However, F. mosseae and G. ver-

MYCORRHIZAL INOCULATION AFFECTS METALS IN PAKCHOI

19

Fig. 3 Mycorrhizal colonization (a), mycorrhizal dependence (b), glomalin-related soil protein (GRSP) concentration (c), and polyphosphate concentration (d) of three arbuscular mycorrhizal fungi (Funneliformis mosseae, Glomus versiforme, and Rhizophagus intraradices) inocula in the rhizosphere of pakchoi after 70 d under different Cd addition treatments. Cd0 = no Cd addition; Cd1 = Cd addition at 0.9 mg kg−1 ; Cd2 = 1.5 mg kg−1 . Values are means with standard deviations shown by the vertical bars (n = 6). Bars with the same letter(s) indicate no significant differences at P < 0.05.

siforme increased Cd concentrations in the roots of pakchois compared with the non-inoculated pakchois in the treatment without Cd addition. Meanwhile, we also found TF of Pb or Cd increased significantly with increasing Pb or Cd addition levels, while the effects of AMF inocula on TF were not significant at each Pb or Cd addition level. Changes of soil properties and metal content Compared to the non-inoculated treatments, the three AMF inocula significantly increased soil pH and EC in Pb2 and Cd2 treatments, but had no significant effects on the total Cd and Pb concentrations at the end of the 70-d experiment (Table VI). Bioavailable Pb or Cd in the AMF inoculated soil declined after 70 d, while the SOM-Pb or Cd concentrations increased compared to the non-inoculated treatments. The concentrations of GRSP-Cd or Pb in the soil had no significant differences among the three AMF inocula. DISCUSSION Brassicaceae family species are generally believed to be non-mycotrophic plants. Previously, some researchers found that AMF can colonize distinctly the

root of some Brassicaceae plants such as cabbage (Brassica oleracea var. Capitata) with G. aggregatum and G. fasciculatum (Nelson and Achar, 2001), pennycress (Thlaspi spp.) with R. intraradices (Regvar et al., 2003) even in heavy metal-polluted soil (Pongrac et al., 2009), and mycorrhizal rape (Brassica napus L.) with F. mosseae and G. versiforme (Li, 2007). In this study, our findings provided the first evidence of mycorrhizal colonization (F. mosseae, G. versiforme, and R. intraradices) in pakchoi cultivated in Pb or Cd-contaminated soils, and heavy metal stress had a strong effect on AMF development. Arbuscular mycorrhizal fungi could develop various different physiological adaptations in response to a direct or indirect toxic effect of heavy metal, which enable them to compete successfully with the harsh conditions in heavy metal-polluted soils (Hildebrandt et al., 2007). All mycorrhizal samples were successfully colonized by AMF on the bases of the occurrence of vesicles, intercellular hyphae, and arbuscular (Fig. 1). Mycorrhizal colonization was significantly greater for G. versiforme than F. mosseae and R. intraradices in Pb addition treatments (Fig. 2a), while that for G. versiforme was lower than the other two

−M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices −M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices −M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices

Pb0

MCR

2 376.9*** 170.1*** 40.5***

39.03 30.26 39.79

35.7 20.39 33.3

20.94 33.86 12.2

%

812.7*** 11.2*** 9.8***

µg pot−1 DW 21.21 ± 0.95ab 20.12 ± 0.98b 22.72 ± 0.62a 21.42 ± 0.39ab 22.73 ± 0.32c 24.32 ± 0.91ab 23.62 ± 0.77bc 25.72 ± 0.89a 32.52 ± 0.91b 34.33 ± 0.75a 31.21 ± 0.77b 35.18 ± 0.52a

Pb concentration

DWc) 0.19d) de) 0.21b 0.12a 0.22c 0.21c 5.28a 1.93b 5.33a 1.50c 3.86a 3.15b 9.02a

Pb concentration µg pot−1 23.11 ± 29.23 ± 34.94 ± 26.32 ± 74.21 ± 115.41 ± 93.22 ± 111.26 ± 91.83 ± 150.62 ± 131.67 ± 152.51 ±

Shoot

Root

5.27 −4.2 7.57

6.54 3.77 11.63

−5.42 6.65 0.98

%

MCR

***Significant at P < 0.001. a) Pb0 = no Pb addition; Pb1 = Pb addition at 125 mg kg−1 ; Pb2 = Pb addition at 250 mg kg−1 . b) −M = non-inoculated. c) Dry weight. d) Means ± standard deviations (n = 6). e) Means followed by the same letter(s) within a column for each Pb addition treatment are not significantly different at P < 0.05. f) Pb concentration in root/Pb concentration in shoot.

F value Pb addition level (LEpb ) AMF inocula (AMFI) LEpb × AMFI

Pb2

Pb1

AMF inoculumb)

Treatmenta)

DW 1.05d 0.98b 0.73a 0.21c 0.27c 4.59a 2.81b 3.82a 2.20c 4.61a 3.11b 6.03a 4 128.7*** 261.6*** 65.1***

µg pot−1 44.32 ± 49.35 ± 57.66 ± 47.74 ± 96.79 ± 139.73 ± 116.84 ± 136.98 ± 124.35 ± 184.95 ± 162.88 ± 187.69 ±

Pb concentration

Total plant

32.77 23.66 33.75

30.62 17.03 29.23

10.19 23.14 7.16

%

MCR

821.9*** 63.7*** 11.1***

1.09 1.45 1.54 1.23 3.27 4.75 3.95 4.33 2.82 4.39 4.22 4.34

Distribution ratiof)

Mycorrhizal contribution rate (MCR) of three arbuscular mycorrhizal fungi (AMF) inocula to Pb accumulation and distribution in roots and shoots of pakchoi under different Pb addition treatments

TABLE III

20 Z. P. WU et al.

−M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices −M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices −M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices

Cd0

55 638.7*** 1 768.1*** 563.3***

15.53 7.29 33.20

23.92 18.64 39.31

29.86 47.70 15.43

5 631.3*** 50.7*** 13.7***

µg pot−1 DW 1.21 ± 0.05c 1.38 ± 0.05b 1.79 ± 0.06a 1.43 ± 0.04b 3.33 ± 0.12b 3.62 ± 0.12a 3.71 ± 0.16a 3.74 ± 0.15a 5.33 ± 0.09c 5.76 ± 0.09b 5.73 ± 0.09b 6.32 ± 0.11a

Cd concentration

MCR %

Cd concentration µg pot−1 DWc) 1.48 ± 0.04d) de) 2.11 ± 0.07b 2.83 ± 0.04a 1.75 ± 0.04c 6.33 ± 0.11d 9.32 ± 0.07b 8.78 ± 0.08c 11.34 ± 0.27a 16.32 ± 0.11d 19.32 ± 0.09b 18.51 ± 0.24c 24.43 ± 0.14a

Shoot

Root

7.47 −3.90 15.67

8.01 −3.74 5.93

12.32 32.40 15.39

%

MCR

***Significant at P < 0.001. a) Cd0 = no Cd addition; Cd1 = Cd addition at 0.9 mg kg−1 ; Cd2 = Cd addition at 1.5 mg kg−1 . b) −M = non-inoculated. c) Dry weight. d) Means ± standard deviations (n = 6). e) Means followed by the same letter within a column for each Cd addition treatment are not significantly different at P < 0.05. f) Cd concentration in root/Cd concentration in shoot.

F value Cd addition level (LECd ) AMF inocula (AMFI) LECd × AMFI

Cd2

Cd1

AMF inoculumb)

Treatmenta)

26 145.3*** 749.4*** 265.3***

µg pot−1 DW 2.69 ± 0.05d 3.49 ± 0.10b 4.62 ± 0.05a 3.18 ± 0.07c 9.66 ± 0.21d 12.94 ± 0.16b 11.99 ± 0.52c 14.97 ± 0.18a 21.65 ± 0.12d 26.08 ± 0.46b 23.64 ± 0.15c 31.75 ± 0.24a

Cd concentration

Total plant

13.7 4.4 29.6

19.1 12.1 30.9

22.9 41.8 15.4

%

MCR

2 396.5*** 130.3*** 49.1***

1.22b 1.53a 1.58a 1.22b 1.91d 2.58b 2.37c 3.06a 3.06c 3.35b 3.23b 3.87a

Distribution ratiof)

Mycorrhizal contribution (MCR) of three arbuscular mycorrhizal fungi (AMF) inocula to Cd accumulation and translocation in roots and shoots of pakchoi under different Cd addition treatments

TABLE IV

MYCORRHIZAL INOCULATION AFFECTS METALS IN PAKCHOI 21

−M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices −M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices −M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices

HM0

± ± ± ± ± ± ± ± ± ± ± ±

kg−1

mg 0.0021d) bce) 0.0012a 0.0009b 0.0004bc 0.0051a 0.0078c 0.0092b 0.0056b 0.0064a 0.0118b 0.0063b 0.0054b

12 1623.3*** 715.9*** 194.9***

0.0372 0.0389 0.0368 0.0374 0.4231 0.2648 0.2977 0.3047 1.3350 1.1309 1.1687 1.1589

Croot

Pb

± ± ± ± ± ± ± ± ± ± ± ± 0.0005b 0.0005a 0.0008b 0.0007b 0.0038a 0.0016c 0.0016b 0.1453b 0.0091a 0.0089b 0.0108b 0.0101b

2 973.9*** 46.8*** 13.5***

0.0294 0.0314 0.0291 0.0297 0.1343 0.0824 0.0935 0.0963 0.2343 0.1938 0.2052 0.2031

FWc)

Cshoot

4 524.1*** 0.06nsf) 0.36ns

0.791a 0.807a 0.791a 0.794a 0.316a 0.311a 0.314a 0.316a 0.175a 0.172a 0.176a 0.176a

TF ± ± ± ± ± ± ± ± ± ± ± ± 2 973.9*** 46.8*** 13.5***

0.00655 0.00703 0.00693 0.00654 0.46845 0.32677 0.32394 0.31320 1.20121 1.03398 1.06192 0.96091

Croot

Cd

mg 0.00021b 0.00012a 0.00019a 0.00064b 0.01112a 0.00512b 0.01021b 0.00632b 0.00723a 0.00412c 0.00632b 0.11821d

± ± ± ± ± ± ± ± ± ± ± ± 458.9*** 9.3*** 2.6*

FW 0.00523 0.00564 0.00551 0.00522 0.14322 0.10112 0.10209 0.09232 0.21243 0.18109 0.18586 0.15671

Cshoot kg−1

0.0006b 0.00023a 0.00034a 0.00067b 0.00626a 0.00427ab 0.04702ab 0.00051b 0.00421a 0.00789b 0.00741b 0.01233c

632.1*** 0.105ns 0.046ns

0.799a 0.802a 0.795a 0.798a 0.306a 0.307a 0.311a 0.295a 0.171a 0.176a 0.176a 0.164a

TF

a) HM

*, ***Significant at P < 0.05 and P < 0.001, respectively. = heavy metal, representing Pb or Cd addition here: Pb0 = no Pb addition; Pb1 = Pb addition at 125 mg kg−1 ; Pb2 = 250 mg kg−1 ; Cd0 = no Cd addition; Cd1 = Cd addition at 0.9 mg kg−1 ; and Cd2 = 1.5 mg kg−1 . b) −M = non-inoculated. c) Fresh weight. d) Means ± standard deviations (n = 6). e) Means followed by the same letter(s) within a column for each Pb or Cd addition treatment are not significantly different at P < 0.05. f) Not significant.

F value HM addition level (LEHM ) AMF inocula (AMFI) LEHM × AMFI

HM2

HM1

AMF inoculumb)

Treatmenta)

Translocation factor (TF) and concentrations of Pb or Cd in roots (Croot ) and shoots (Cshoot ) of pakchois inoculated with three arbuscular mycorrhizal fungi (AMF) under different Pb or Cd addition treatments

TABLE V

22 Z. P. WU et al.

MYCORRHIZAL INOCULATION AFFECTS METALS IN PAKCHOI

23

TABLE VI Selected propertiesa) of the soils planted with pakchoi inoculated with arbuscular mycorrhizal fungi (AMF) after 70 d in the treatments of Pb addition at 250 mg kg−1 (Pb2) or Cd addition at 1.5 mg kg−1 (Cd2) Treatment AMF inoculumb)

pH

EC

Total Pb

Bioavailable Pb GRSP-Pb

cm−1

F value

6.53 ± 0.01c) cd) 6.68 ± 0.04b 6.86 ± 0.05a 6.81 ± 0.02a 21.62***

µS 46.3 ± 47.2 ± 48.1 ± 47.8 ± 5.40*

Treatment AMF inoculum

pH

EC

Pb2

−M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices

0.4b 0.6ab 0.8a 0.5a

262.22 ± 259.23 ± 265.11 ± 260.01 ± 1.01nse)

5.21a 4.54a 4.69a 4.55a

Total Cd

cm−1

Cd2

F value

−M Funneliformis mosseae Glomus versiforme Rhizophagus intraradices

6.55 ± 0.06b 6.63 ± 0.05b 6.83 ± 0.04a 6.79 ± 0.04a 23.03***

µS 45.9 ± 47.8 ± 46.2 ± 47.5 ± 4.31*

0.5c 0.7a 1.0bc 0.9ab

1.54 ± 1.61 ± 1.56 ± 1.59 ± 0.64ns

0.11a 0.05a 0.02a 0.05a

40.17 ± 36.84 ± 37.45 ± 38.13 ± 14.91**

µg 1.12a 0.39c 0.29bc 0.45b

– 54.02 ± 0.26d 420.53 ± 13.01a 58.34 ± 0.47b 421.14 ± 9.21a 59.12 ± 0.47a 451.18 ± 17.98a 57.32 ± 0.20c 4.79ns 110.49**

Bioavailable Cd GRSP-Cd µg 0.18 ± 0.01a 0.12 ± 0.02bc 0.11 ± 0.02c 0.15 ± 0.02ab 10.91**

SOM-Pb

g−1

SOM-Cd

g−1 – 1.05 ± 0.04a 1.03 ± 0.05a 1.06 ± 0.03a 0.46ns

0.23 ± 0.28 ± 0.31 ± 0.25 ± 5.65*

0.03b 0.02ab 0.02a 0.04b

*, **, ***Significant at P < 0.05, P < 0.01 and P < 0.001, respectively. a) EC = electrical conductivity; GRSP = glomalin-related soil protein; SOM = soil organic matter. b) −M = non-inoculated. c) Means ± standard deviations (n = 6). d) Means followed by the same letter(s) within a column for Pb2 or Cd2 treatment are not significantly different at P < 0.05. e) Not significant.

inocula in Cd addition treatments (Fig. 3a). The results suggested the fungi symbiosis was not only in response to AMF inocula, but closely involved in the metal behavior in soil-plant system (Hildebrandt et al., 2007). Similarly, some researches showed the mycorrhizal colonization rates of plants varied from AMF inocula to inocula such as mycorrhizal Zea mays L. grown in the As-contaminated soil (Yu et al., 2010) and inoculated Astragalus sinicus L. in the high Cd-contaminated soil (Li et al., 2009). However, Chen et al. (2004) found that Cd contamination does not affect the mycorrhizal colonization of Zea mays L. by F. mosseae. Additionally, inoculation effectiveness was reflected in plant growth traits and MD under different environmental conditions (Yang et al., 2011). We observed that all AMF inocula significantly increased biomass of pakchoi shoots at over 8.7% in all heavy metal addition treatments compared to the corresponding non-inoculated treatments (Figs. 2b and 3b). In our study, F. mosseae, G. versiforme, and R. intraradices significantly decreased Pb or Cd concentrations in pakchoi shoots and roots in the soils with Pb or Cd addition compared to the non-inoculated plants (Table V). Similarly, the negative effects of AMF inocula on Cd acquisitions were found in bashfulgrass inoculated with either G. caledonium or G. versiforme (Hu et al., 2014), maize with F. mosseae (Chen et al., 2004), and marigold (Tagetes erecta L.) with R. intraradices or F. mosseae (Liu et al., 2011). Gu et al. (2013) found F. mosseae inoculation substantial-

ly enhanced the growth of F. arundinacea and significantly decreased Pb and Cd concentrations in shoots in a greenhouse experiment. Meanwhile, we also observed that Pb concentrations in pakchoi shoots were decreased significantly by 20.6%–67.5% in the Pb addition soils by the three AMF inocula, which were all below the allowable limit of Chinese Food Safety Standard of China (GB2762-2005). The Cd concentrations were also significantly reduced (14.3%–54.1%) by the three AMF inocula relative to the non-inoculated plants, which were less than the maximum allowable limit of 0.2 mg kg−1 . Thus, it is worth emphasizing that AMF inoculation would be an optional strategy to enhance the yield and reduce the food safety risk of vegetable grown on Pb or Cd-contaminated soils, though there were significant differences in MCR, MD, and metal acquisition among the three AM fungi inocula. Actually, AMF have repeatedly been demonstrated to alleviate heavy metal stress of plants in heavy metal-contaminated soils via some mechanisms, such as the retention of toxic metals in roots and reduction of translo cation to shoots (Tullio et al., 2003; Deng et al., 2004; Janouˇskov´a et al., 2005). Our results showed that AMF significantly decreased the Pb or Cd concentrations in pakchoi roots and shoots in the Pb or Cd addition soils, and increased metal concentrations in pakchois cultivated in no Pb or Cd addition (Table V), emphasizing that AMF might have the stress tolerance potential, especially in heavy metal-contaminated soils

Z. P. WU et al.

24

(Hildebrandt et al., 2007). We also found that AMF significantly enhanced the total Pb or Cd amounts and the distribution rate (root/shoot) independent of the soil Pb or Cd concentrations (except for a few samples), but did not significantly alter the translocation efficiencies of Pb or Cd by pakchoi (Table IV). The positive growth response to AM symbiosis may also account for the phenomena due to the biomass dilution effect (Janouˇskov´ a et al., 2007). Another detoxification mechanism of mycorrhizal plant is the ability of AMF to bind heavy metals by fungal hyphae (hyphae wall, inner chambers, and the vacuole) (Zhang et al., 2010), as well as the sequestration of metals by glomalin excreted by AMF (Gonz´alez-Ch´ avez et al., 2004; Wu et al., 2014) in line with our findings. In addition, we observed that free polyphosphate concentrations detected by TBO in the hyphae of mycorrhizal plant were significantly reduced with the rising of soil Pb or Cd levels, which suggested that heavy metal ions were sequestered or hydrolyzed by polyphosphate in order to resist heavy metal stress (Gonzalez and Jensen, 1998; Seufferheld et al., 2008). Mycorrhizal roots can change the rhizospheric soil environment, thereby influencing heavy metal forms associated with heavy metal behavior in soil-plant system (Huang et al., 2000). Soil DTPA-extractable metals stands for the bioavailable fraction of metals in soils. It is clear that compared with the non-inoculated soils, the three AMF inocula significantly decreased soil bioavailable Pb or Cd fraction, while significantly increased the SOM-Pb or Cd fraction after plant harvest (Table VI). The results could be attributed to AMF inoculation altering root secretion composition and pH in the rhizospheric soil (Kirk and Bajita, 1995). In fact, soil pH is commonly served as an important factor for metal bioavailability (Zhao et al., 2003). We found that AMF inocula increased soil pH compared to the non-inoculated treatments, which may be resulted from HCO3− (OH− ) exudation via the excess uptake of anions and/or active nitrate uptake (Bago et al., 1996; Marschner and Marschner, 2012) or the reduction to secrete H+ (Degenhardt et al., 1998) by AMF inoculation. Thus, the protective benefit of mycorrhizal plant exposed to Pb or Cd stress may be related to the decrease of soil metal phytoavailability by increasing soil pH. Furthermore, the uptake and accumulation of metal in host plants could be affected by AMF isolated from habitats contaminated with heavy metals or noncontaminated soils (Hildebrandt et al., 2007; Liu et al., 2011). The colonization rate of AMF isolates also shows differently when present in a mixed AMF

community or experimental conditions (Jacquot et al., 2000; Li et al., 2009). Therefore, it is necessary to apply mycorrhizal seedlings into a field experiment containing indigenous AMF in future studies. CONCLUSIONS Different effects of AMF inocula, F. mosseae, G. versiforme, and R. intraradices, on mycorrhizal dependence and polyphosphate and GRSP concentrations were observed depending on species of fungal symbiont. The AMF inocula also significantly decreased plant Pb and Cd concentrations and had no significant effects on Pb and Cd translocation efficiencies in the soils with Pb or Cd addition, though they increased the total soil metal concentrations compared to the noninoculated treatments. Meanwhile, AMF decreased the phytoavailability of Pb and Cd by increasing soil pH and EC. Therefore, AMF inocula had positive effects on the growth and development of pakchoi in heavy metal-contaminated soils. Our results provided a new perspective of the role of AMF in promoting safe vegetable production. ACKNOWLEDGEMENTS This study was financially supported by the Ministry of Agriculture of China as an industry special project (No. 200903015) and the National Natural Science Foundation of China (No. 41001047). We are indebted to all of the staff at the cell biology lab in the Analytical and Testing Center of Nanjing University, China for the analysis of the soil samples. REFERENCES Akkajit P, Tongcumpou C. 2010. Fractionation of metals in cadmium contaminated soil: Relation and effect on bioavailable cadmium. Geoderma. 156: 126–132. Bago B, Vierheilig H, Pich´ e Y, Azc´ on-Aguilar C. 1996. Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture. New Phytol. 133: 273–280. Cartea M E, Francisco M, Soengas P, Velasco P. 2010. Phenolic compounds in Brassica vegetables. Molecules. 16: 251–280. Chen B D, Liu Y, Shen H, Li X L, Christie P. 2004. Uptake of cadmium from an experimentally contaminated calcareous soil by arbuscular mycorrhizal maize (Zea mays L.). Mycorrhiza. 14: 347–354. Degenhardt J, Larsen P B, Howell S H, Kochian L V. 1998. Aluminum Resistance in the Arabidopsis Mutant alr-104 is caused by an aluminum-induced increase in rhizosphere pH. Plant Physiol. 117: 19–27. Deng H, Ye Z H, Wong M H. 2004. Accumulation of lead, zinc, copper and cadmium by 12 wetland plant species thriving in metal-contaminated sites in China. Environ Pollut. 132: 29–40.

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