Ecotoxicology (2014) 23:1979–1986 DOI 10.1007/s10646-014-1331-6
Growth, cadmium uptake and accumulation of maize (Zea mays L.) under the effects of arbuscular mycorrhizal fungi Lingzhi Liu • Zongqiang Gong • Yulong Zhang Peijun Li
•
Accepted: 9 August 2014 / Published online: 5 September 2014 Ó Springer Science+Business Media New York 2014
Abstract The effects of three arbuscular mycorrhizal fungi isolates on Cd uptake and accumulation by maize (Zea mays L.) were investigated in a planted pot experiment. Plants were inoculated with Glomus intraradices, Glomus constrictum and Glomus mosseae at three different Cd concentrations. The results showed that root colonization increased with Cd addition during a 6-week growth period, however, the fungal density on roots decreased after 9-week growth in the treatments with G. constrictum and G. mosseae isolates. The percentage of mycorrhizal colonization by the three arbuscular mycorrhizal fungi isolates ranged from 22.7 to 72.3 %. Arbuscular mycorrhizal fungi inoculations decreased maize biomass especially during the first 6-week growth before Cd addition, and this inhibitory effect was less significant with Cd addition and growth time. Cd concentrations and uptake in maize plants increased with arbuscular mycorrhizal fungi colonization at low Cd concentration (0.02 mM): nonetheless, it decreased at high Cd concentration (0.20 mM) after 6-week growth period. Inoculation with G. constrictum isolates enhanced the root Cd concentrations and uptake, but G. mosseae isolates showed the opposite results at high Cd concentration level after 9 week growth period, as compared to non-mycorrhizal plants. In conclusion, maize plants inoculated with arbuscular mycorrhizal fungi were less sensitive to Cd stress than uninoculated plants. G.
L. Liu (&) Y. Zhang College of Land and Environment, Shenyang Agricultural University, Shenyang 110086, People’s Republic of China e-mail: [email protected] L. Liu Z. Gong P. Li Institute of Applied Ecology, Chinese Academy of Science, Shenyang 110016, People’s Republic of China
constrictum isolates enhanced Cd phytostabilization and G. mosseae isolates reduced Cd uptake in maize (Z. mays L.). Keywords Arbuscular mycorrhiza Zea mays L. Cd accumulation Phytoremediation
Introduction Cadmium (Cd) is a potentially phytotoxic heavy metal (HM). It is released into the environment by anthropogenic activities including mining, industry, and application of sewage sludge to agricultural land, on most occasions resulting in accumulation of Cd in soils (Janouskova´ et al. 2006). Soil pollution by Cd become a critical environmental concern due to its potential adverse ecological effects. High cadmium concentration in the soil has detrimental effects on ecosystem and is a risk to human health due to cadmium accumulation in food chains (Garg and Bhandari 2012; Glassman and Casper 2012). Cd is not biodegradable, remediation of Cd contaminated soil is challenging due to its unique properties. The use of physical or chemical methods for this purpose is often not cost effective, thus phytoremediation is becoming an increasingly important technology in soil remediation by offering an inexpensive and sustainable approach based on the immobilization of Cd within roots (phytostabilization) or to import and store Cd in the plant’s above-ground tissues (phytoextration) (Rashid et al. 2009). However, phytoremediation is a slow process and it is necessary to improve the efficiency to increase the stabilization or removal of Cd from soils. Arbuscular mycorrhizal fungi (AMF) provide an attractive system to advance phytostabilization of Cd by compounds secreted by the fungus, precipitation in polyphosphate granules in the soil,
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adsorption to fungal cell walls, and chelation of Cd inside the fungus (Glassman and Casper 2012; Jankong and Visoottiviseth 2008; Tang et al. 2009). Therefore, the use of mycorrhizal plants for land remediation has been proposed (Zarei et al. 2010; Carrasco et al. 2011; Go¨hre and Paszkowski 2006) with results such as mycorrhizae influencing metal transfer in plants by increasing plant biomass and plant phosphorus nutrition (Shen et al. 2006; Liu et al. 2011), reducing metal toxicity to plants by decreasing root to shoot HM translocation and shoot HM concentrations (Paul-Olivier et al. 2008). Many results showed that several factors such as AMF isolates, plant species, soil contamination levels and soil characteristics could determine the symbiotic conditions between the plants and the AMF isolates (Veresoglou et al. 2012). The effect of AMF on the uptake of Cd by plants is different with different reports. Among these researches, much attention has been paid to the influence of a single AMF isolate in the uptake and accumulation of metals by plants, while whether the similar AMF species would showed the same results is not yet totally clear. To use the arbuscular mycorrhiza (AM) symbiosis for phytoremediation, it is important to understand the compatibility between host plants and AMF isolates under the Cd contaminated conditions. Examination of the effect of Cd on symbiotic fungal life stages of two Glomus species showed Glomus intraradices to be more tolerant to Cd than Glomus etunicatum (Pawlowska and Charvat 2004). Zhang et al. (2005) reported that among the three tested Glomus species, Glomus mosseae inoculation gave the most protective effects on the upland rice under the combined soil contamination. These results showed that selection a compatible AMF isolate and plant may be useful for phytoremediation. Maize is a high biomass crops with increased ability to tolerate and accumulate Cd by introduction of metalbinding proteins or peptides and therefore is considered and tested as an alternative. However, studies of maize plants with different AMF isolates showed different results. Chen et al. (2004) and Liang et al. (2009) reported that inoculation with G. mosseae could enhance the biomass of maize plants and reduce the Cd translocation from root to shoot by immobilizating much Cd in roots. However, other studies showed that Glomus caledonium had no significant or negative effect on maize biomass and sent more Cd to shoots (Liao et al. 2003; Wang et al. 2007a, b), and Shen et al. (2006) showed that Cd addition level would influence the mycorrhizal effect. Therefore, the symbiotic effect of Glomus species and maize plant at different Cd addition level still needs to further study to meet with the phytoremediatione. The objective of this study is to investigate whether AM could decrease the Cd uptake and accumulation in maize
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plants under different Cd addition level. Three AMF, G. intraradices (GI), Glomus constrictum (GC) and G. mosseae (GM) were selected to colonize maize plants.
Materials and methods Plant, soil and AMF inoculum AM fungal inoculums used in the experiment consisted of G. intraradices (BGC USA05), G. constrictum (BGC USA02) and G. mosseae (BGC NM04A). The three AMF isolates were propagated in the laboratory in pot cultures during 4 months’ cycles with sorghum seedlings growing in pots containing a 1:1 (v/v) mixture of soil and sand. Inoculums containing rhizosphere soil, root, mycelium and spores of the three AMF isolates were applied to Zea mays growing in soils at different Cd levels, respectively. The soil used in the experiment was collected from Shenyang agriculture university, Liaoning Province, North China, with the following properties: pH (in H2O) 6.0, available K 20.26 mg kg-1, total C 2.83 %, total N 0.43 g kg-1, available P 14.84 mg kg-1. The soil was mixed with washed river sand (sand/soil, 1:2 v/v). Experimental design Seeds of Z. mays L.were surface sterilized with 10 % (v/v) hydrogen peroxide for 15 min, rinsed with sterile distilled water for 5–6 times. Sterilized seeds were germinated on Petri dishes lined with moisturized filter paper and placed in the dark at 25 °C for 2–3 days. Uniform seedlings were then selected and transferred to plastic containers (13 cm diameter, and 13 cm height) containing washed river sand (\2 mm) autoclaved at 121 °C for 1 h on two consecutive days. The experimental pots were disinfected with 70 % ethanol before soil was loaded. Each pot was first filled with 1.0 kg prepared mixture of soil and sand, then 50 g of inoculums was spread over the surface; for the nonmycorrhizae control the inoculums had been autoclaved. Finally the pots were covered with 0.25 kg prepared mixture of soil and sand. Four replicates were performed for individual treatments. The plants grew in a controlled environmental growth room, with temperatures of 25/18 °C (day/night), a photoperiod of 14 h at a quantum flux density of 400 lmol m-2 s-1 and 50 % relative humidity. The plants were watered with tap water as necessary and received 1/4-strength Hoagland modified solution, with a reduced P concentration (0.1 mM) once every 2 weeks. The pots were in a completely randomized design and were randomly rotated at weekly intervals to avoid site effects within the growth room.
Contribution of arbuscular mycorrhizal fungi in soil remediation
80
a
a
a
60
ab
a
b b
b
40 c
20
b Root colonization (%)
a Root colonization (%)
Fig. 1 Effects of cadmium addition on Zea mays L. root colonization by AMF (a 6 weeks, b 9 weeks). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
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20 0
No Cd
0.02 mM
0.2 mM
No Cd
0.02 mM
0.2 mM
Cd addition level GI
After the inoculation period, maize plants were to Cd addition. There were three AMF inoculum (GI, GC and GM inoculums) and four Cd levels (0, 0.02, 0.20 mM) with non-mycorrhizal control (NM). Forty milliliters of 1/4strength Hoagland modified solution with a reduced P concentration (0.1 mM) and with three different concentrations of Cd was added as CdCl2 weekly to each corresponding pots.
GC
GM
were performed using Duncan’s multiple range test (P \ 0.05). Prior to statistical analysis, data were logarithmically transformed [y = ln(x ? 1)] to satisfy the assumptions of the ANOVA. Software SPSS 11.5 was used for all the statistical analyses. Sigmaplot 10.0 for Cartography.
Results Harvest and analyses Mycorrhizal colonization Plants were harvested after 6- and 9-week, respectively. Shoots were first cut off, and roots were collected, washed gently with tap water, and then rinsed in deionized water. The clean roots were cut into segments about 1 cm long. A randomly selected subsample of one fresh root (0.2 mg) was taken for assessment of root colonization. Percentages of root colonization by the AMF were estimated by the gridline intersection method at 40–1609 magnification after clearing and staining the roots with trypan blue in lactoglycerol (Koske and Gemma 1989). Biomasses of the plant roots and shoots were determined after drying at 70 °C for 3 days. For the analysis of Cd concentrations in the root and shoot samples, the plant samples were ground, digested by HNO3 and HClO4 (3:1), and the Cd concentrations were determined by atomic absorption spectrometry with flameless atomization. Total Cd (accumulation) in the whole plant was calculated by the following equation: Total Cd = shoot Cd concentration (lg g-1) 9 shoot biomass (g) ? root Cd concentration (lg g-1) 9 root biomass (g). Statistical analysis Data were analyzed with a two-way analysis of variance (ANOVA) to assess the effects of AMF inoculation and cd levels in the experiment, for percentage of root length colonization, the NM control was excluded from the analysis due to no colonization. Comparisons between means
No root colonization was found in the NM control before the onset and after the treatment. Root colonization increased with increasing Cd addition except for a slight decrease for GC treatment at high Cd addition (Fig. 1a). Root colonization was significantly influenced by AMF inoculation (F = 39.21, P \ 0.001), Cd addition level (F = 35.7, P \ 0.001), and the interaction between AMF and Cd concentrations (F = 20.47, P \ 0.001). Cadmium addition (0.20 mM) inhibited root colonization in the GM and GC treatments (Fig. 1b). Generally, the percentage of colonization in mycorrhizal maize ranged from 30.3 to 72.3 % under different Cd concentrations. Among the three isolates, GI inoculation showed the highest root colonization as compared with GM and GC treatments. Plant biomass Cd decreased plant growth in the NM control treatment. Nonetheless, mycorrhizal colonization sustained the shoot and root biomass during the first 6-week growth period without Cd addition (Fig. 2a; Table 1). However, this inhibitory effect got less significant for shoot biomass after 9-week growth (Table 1) and GC and GM inoculation significantly increased the plant biomass (P \ 0.05) at 0.02 mM Cd addition level (Fig. 2b), compared with the NM control. Among the three AMF isolates, the biomasses of Z. mays L. did not differ significantly between
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a
10 8
b
a b
14
b
bc
bc
b
b bc
c
6 4 2
a
a
12
b b bc
Total biomass (g)
Fig. 2 Effects of cadmium addition and AMF inoculation on biomass of Zea mays L (a 6 weeks, b 9 weeks). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
Total biomass (g)
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10
a b
b
b
b
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b
b
8 6 4 2
0
0 0
0.02 mM
0.20 mM
0
0.02 mM
0.20 mM
Cd addition level GI
Table 1 Effect of different AMF on the growth of Zea mays L. under Cd stress
Cd addition level (mM)
Root to shoot ratio
6 week
9 week
6 week
9 week
6 week
9 week
GI
4.9b
6.42b
1.86cd
3.52 fg
0.38d
0.55def
GC
4.88b
6.32b
1.91cd
3.88de
0.39d
0.61bcd
GM
4.61bc
6.39b
1.81d
3.25 g
0.39d
0.51f
NM
5.88a
6.24b
2.78a
5.13a
0.47b
0.82a
GI
4.12c
6.40b
1.75d
3.72ef
0.43bcd
0.58cdef
GC
4.53bc
7.51a
1.87cd
3.95cde
0.42bcd
0.53ef
GM
4.84b
7.25a
2.03bc
4.30b
0.42cd
0.59cde
NM GI
4.61bc 4.57bc
6.16b 5.98b
2.14bc 1.80d
4.19bc 4.07bcd
0.47bc 0.39d
0.68b 0.69b
GC
4.78b
6.42b
1.90cd
3.41g
0.40 cd
0.53ef
GM
4.13c
6.38b
2.22b
3.80def
0.54a
0.60cde
NM
4.7bc
6.00b
2.05bc
3.83de
0.44bcd
0.64bc
Mycorrhiza
**
***
***
***
**
***
Cd level
***
***
n.s.
**
n.s.
n.s.
Mycorrhiza 9 Cd level
**
*
***
***
**
***
Different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
0.20
a
Significancea due to:
n.s. non-significant effect
treatments under the same Cd addition level, except for 0.02 mM of Cd addition at 9-week growth. Plant Cd concentrations Cd addition level and AMF inoculation significant influenced Cd concentrations in the shoots (P \ 0.01) and roots (P \ 0.01) of maize. After 6-week treatment period, maize with AMF inoculation had higher Cd concentrations in the shoot and root than that in the non-inoculated treatment at low Cd addition level (Fig. 3a, b). However, this effect became not significant or even a metal uptake decrease appeared on shoot biomass after 9-week growth at this Cd addition level. In the treatment with 0.2 mM Cd, mycorrhizal colonization decreased Cd concentration in maize slightly after 6-week growth compared to the non-inoculated treatment (Fig. 3c, d). However, effects of the three AMF isolates on Cd concentrations in the root of maize
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NM
Root biomass (g)
0.02
Significant effects according to two-way ANOVA: * P \ 0.05; ** P \ 0.01; *** P \ 0.001
GM
Shoot biomass (g)
0
Inoculation type
GC
were different after 9-week growth, GC and GM inoculation significantly enhanced and decreased Cd concentration (P \ 0.05), respectively, while GI inoculation showed no effect on the Cd concentration. Plant Cd uptake and accumulation At low Cd addition level, AMF inoculation enhanced total Cd uptake in the plants in the first harvest, however the AMF inoculation reduced Cd uptake in the final harvest. With increasing Cd addition level, AMF inoculation significantly decreased Cd uptake in the plants during 6-week growths. Inoculation with GC, GM and GI isolates significantly increased (P \ 0.05), decreased (P \ 0.05) and had no effect on the Cd uptake in the plants, compared with that in the non-inoculated plants (Fig. 4a, b). The ratio of Cd in the roots to the shoots showed a large differences in the three AMF inoculations as compared to
Contribution of arbuscular mycorrhizal fungi in soil remediation
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-1
Root concentration (mg kg )
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Root concentration (mg kg )
-1
Shoot concentration (mg kg )
a
Shoot concentration (mg kg )
Fig. 3 Effects of cadmium addition and AMF inoculation on Cd concentration of root and shoot of Zea mays L. (a, b 0.02 mM Cd, c, d 0.2 mM Cd). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
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bc c
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b
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c
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0
9w
6w
9w
Harvest time GC
GI
a
250
b
NM
800 b
200
Total Cd uptake (ug)
a
Total Cd uptake (ug)
Fig. 4 Effects of cadmium addition and AMF inoculation on Cd accumulation in Zea mays L (a 6 weeks, b 9 weeks). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
GM
b c c
150 a b
ab b
0 0.02mM
600
a
b
c
b
ab
a
c
0
0.20mM
0.02 mM
0.20 mM
Cd addition level GI
the NM treatments (P \ 0.05) at high Cd concentration in 9-week growths (Table 2). The higher root/shoot ratio indicated that plants with GC and GM inoculation Tran located less Cd from root to shoot than the NM treatment, and Cd uptake decreased by 83.1 and 68.0 %, respectively compared with the NM control. Although GM isolates increased Cd allocation to roots, the root Cd uptake in the plants was still 0.81 times to the NM control because of the low total Cd uptake in plants. To GI inoculation, it almost had no difference with NM treatment in Cd uptake and accumulation.
Discussion AMF colonization and plant biomass Studies have shown that high levels of heavy metals can inhibit, or even eliminate AMF colonization (Miransari
GC
GM
NM
2011; Weissenhorn and Leyval 1995). In contrast, some other studies found that the Cd contamination did not affect the root colonization (Chen et al. 2004; de Andrade et al. 2008). In our study, high levels of Cd concentrations had a strong effect on AMF development, and the three isolates exhibited different responses to the Cd, with GC and GM colonization being decreased significantly and GI being increased slightly in 9-week growths. The addition of Cd had no significant effect on colonization by GI isolates during the treatment period, showing that the association between GI isolates and Z. mays L. could be well established in Cd contaminated soils, GI inoculation had no effect on biomass enhancement, Cd uptake and allocation in maize plants, compare to NM treatment. The root colonization of 30–70 % observed in the pot experiment indicated that different level of compatibility between host plants and AMF isolates could occur under the Cd contaminated conditions. Growth time, Cd addition levels, and
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Table 2 Effect of different AMF isolates on the Cd uptake in shoots and roots of Zea mays L. under Cd stress Cd addition level (mM) 0.02
Inoculation type
Shoot Cd uptake (lg)
Root Cd uptake (lg)
Root to shoot Cd uptake ratio
6 week
6 week
6 week
9 week
9 week
9 week
GI
2.44d
3.44d
9.40d
12.37d
3.91b
3.62ab
GC
2.13d
8.27d
7.81d
14.12d
3.71b
1.71ef
GM
2.37d
7.65d
8.85d
16.01d
3.74b
2.10de
NM
1.93d
11.78d
8.11d
14.85d
4.21b
1.29f
GI
48.04b
190.50a
105.33b
465.99b
2.21c
2.49cde
GC
52.47a
157.46b
90.04c
529.17a
1.74c
3.41ab
GM NM
27.26c 56.13a
128.87c 189.47a
142.21a 135.10a
380.22c 470.60b
5.30a 2.42c
2.98bc 2.50cde
Mycorrhiza
***
***
***
**
***
n.s.
Cd level
***
***
***
***
***
***
Mycorrhiza 9 Cd level
***
***
**
***
***
***
0.20
Significancea due to:
Different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05) a
Significant effects according to two-way ANOVA: * P \ 0.05; ** P \ 0.01; *** P \ 0.001
n.s. non-significant effect
AMF isolates used might have affected the symbiotic status between the maize plants and the AMF isolates (Zhang et al. 2005). Previous studies showed that mycorrhizal plants had higher plant biomass compared to non-mycorrhizal plants (Leung et al. 2007, 2010; Xu et al. 2008). Differently, Liao et al. (2003) and Wang et al. (2007a, b) reported a decrease in plant biomass with Mycorrhizal colonization. Our study in the initial harvest was consistent with the latter. Although the inhibition from AMF disappeared gradually with the growth and Cd addition, and an obvious decrease in dry weight occurred in the non-inoculated plants with Cd addition, almost no positive effect of AMF on Z. mays L. resistance to Cd was observed. The growth responses to mycorrhiza might be due to differences in isolates of three AMF, plant species, soil characteristics used in the experiment. (Liao et al. 2003; Zhang et al. 2005). This lack of mycorrhizal effect on plant size is, however, an important requisite to demonstrate mycorrhizal influence on Cd uptake and accumulation without confounding dilution effects. Cd uptake and accumulation In our study, Z. mays L. colonized by AMF, especially at low Cd addition levels, had higher Cd concentrations in both shoots and roots in the first harvest, however, similar and lower Cd concentration in roots and shoots in the final harvest. With increasing Cd addition level, AM colonization reduced Cd concentration in the plants both during 6and 9-week growths. While GC isolates inoculation
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increased root Cd concentration in plants in 9-week growths. These results showed that AMF enhance Cd uptake in plants at low Cd addition levels and restrict Cd translocation from soil to plant at high Cd stress to reduce Cd toxicity to the plants, which is in accordance with the study by Vogel-Mikus et al. (2006), showing that inoculation with AMF decreased Zn and Cd concentrations of plant leaves at high soil metal concentration, and increased metal concentrations of plant leaves at low soil concentration. The effect of AMF on metal uptake in plants is different from other studies reporting that AMF increased uptake with reduced (Huang et al. 2006; Liang et al. 2009) or increased (Citterio et al. 2005) transfer to shoots and decreased uptake (Chen et al. 2003), sometimes even no uptake effects (Guo et al. 1996). Plants inoculated with three AMF isolates, especially in the presence of high Cd levels, had lower Cd uptake in the plants in the first harvest, however showed different results in the second harvest, GC, GM and GI isolates inoculation had higher, lower and the same Cd uptake in the plants than that in the nonmycorrhizal plants, respectively. Since mycorrhizal Z. mays L. did not have higher biomass than non-mycorrhizal Z. mays L. at high Cd addition level, the different results in three AMF isolates inoculation may attribute to different protection mechanism in the resistance of mycorrhizal plants to Cd stress. AM colonization immobilizes HM within the soil or within roots and therefore improves phytostabilization. Gaur (2004) and Gonza´lez et al. (2004) reported that extraradical hyphae from AMF can transport metals from soils to plants, but the transfer from fungus to
Contribution of arbuscular mycorrhizal fungi in soil remediation
plant can be restricted due to immobilization in the root tissues by compounds secreted by the fungus, precipitation in polyphosphate granules in the soil, adsorption to fungal cell walls and chelation of metals inside the fungus. In this study, the higher root to shoot Cd uptake ratio in GC and GM inoculation than NM plants at the high levels of Cd addition suggested that the proper AMF would immobilize Cd in their mycelium or induced metal-binding capacity in Z. mays L. roots, thus relatively less of the Cd could be translocated to the shoot (Tonin et al. 2001; Chen et al. 2004; Luis et al. 2006). With creating a more balanced environment in symbioses, AM colonization seems to improve the roots to cope with higher Cd concentrations ultimately. Among the three AMF isolates, GC isolates showed the potential use in Cd phytostabilization by absorbing more Cd in roots and GM isolates showed the potential in improving food quality by reducing Cd uptake in plants, especially in translocation to shoots. The results from above may be useful in appreciate mycorrhizal symbioses selection for different purposes. In general, the GC and GM isolates decreased the Cd translocation from the roots to shoot in the inoculated maize plants indicating the potential that some AMF may have in stress tolerance in Cd contaminated soils. Thus, by limiting Cd uptake and trapping this toxic element in the mycorrhizal symbiont, GM and GC isolates may contribute to reduce Cd entering the food chain and show a potential in phytostabilization applications. Acknowledgments Financial supports from NSFC (40930739 and 31101504) and Ministry of Environmental Protection of China, and arbuscular mycorrhizal isolates provided by Prof. Y. S. Wang (Beijing Academy of Agriculture and Forestry, China) are gratefully acknowledged. Conflict of interest of interest.
The authors declare that they have no conflict
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1985 de Andrade SA, da Silveira AP, Jorge RA (2008) Cadmium accumulation in sunflower plants influenced by arbuscular mycorrhiza. Int J Phytoremediation 10(1):1–13 Garg N, Bhandari P (2012) Influence of cadmium stress and arbuscular mycorrhizal fungi on nodule senescence in Cajanus cajan (L.) Millsp. Int J Phytoremediation. 14:62–74 Gaur A, Adholeya A (2004) Prospects of arbuscular mycorrhizal fungi in phytoremediation of heavy metal contaminated soils. Current Sci. 86:528–534 Glassman SI, Casper BB (2012) Biotic contexts alter metal sequestration and AMF effects on plant growth in soils polluted with heavy metals. Ecology 93:1550–1559 Go¨hre V, Paszkowski U (2006) Contribution of the arbuscular mycorrhizal symbiosis to heavy metal phytoremediation. Planta 223:1115–1122 Gonza´lez-Cha´vez MC, Carrillo-Gonza´lez R, Wright SF, Nichols KA (2004) The role of glomalin, a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements. Environ Pollut 130(3):317–323 Guo Y, George E, Marschner H (1996) Contribution of an arbuscular mycorrhizal fungus to the uptake of cadmium and nickel in bean and maize plants. Plant Soil 184:195–205 Huang H, Zhang S, Chen BD, Wu N, Shan XQ, Christy P (2006) Uptake of atrazine and cadmium from soil by maize (Zea mays L.) in association with the arbuscular mycorrhizal fungus Glomus etunicatum. J Agric Food Chem 54(25):9377–9382 Jankong P, Visoottiviseth P (2008) Effects of arbuscular mycorrhizal inoculation on plants growing on arsenic contaminated soil. Chemosphere 72:1092–1097 Janouskova´ M, Pavlı´kova´ D, Vosa´tka M (2006) Potential contribution of arbuscular mycorrhiza to cadmium immobilisation in soil. Chemosphere 65:1959–1965 Koske RE, Gemma JN (1989) A modified procedure for staining roots to detect VA mycorrhizas. Mycol Res 92:486–505 Leung HM, Ye ZH, Wong MH (2007) Survival strategies of plants associated with arbuscular mycorrhizal fungi on toxic mine tailings. Chemosphere 66(5):905–915 Leung HM, Wu FY, Cheung KC, Ye ZH, Wong MH (2010) Synergistic effects of arbuscular mycorrhizal fungi and phosphate rock on heavy metal uptake and accumulation by an arsenic hyperaccumulator. J Hazard Mater 181:497–507 Liang CC, Li T, Xiao YP, Liu MJ, Zhang HB, Zhao ZW (2009) Effects of inoculation with arbuscular mycorrhizal fungi on maize grown in multi-metal contaminated soils. Int J Phytoremediation 11(8):692–703 Liao JP, Lin XG, Cao ZH, Shi YQ, Wong MH (2003) Interactions between arbuscular mycorrhizae and heavy metals under sand culture experiment. Chemosphere 50(6):847–853 Liu LZ, Gong ZQ, Zhang YL, Li PJ (2011) Growth, cadmium accumulation and physiology of marigold (Tagetes erecta L.) under the effects of arbuscular mycorrhizal fungi. Pedosphere 21:319–327 Luis MC, Isabel CM, Ame´lia M (2006) Arbuscular mycorrhizal fungi enhance root cadmium and copper accumulation in the roots of the salt marsh plant Aster tripolium L. Plant Soil 285:161–169 Miransari M (2011) Hyperaccumulators, arbuscular mycorrhizal fungi and stress of heavy metals. Biotechnol Adv 29:645–653 Paul-Olivier R, Thierry B, Corinne L (2008) Influence of Glomus intraradices on Cd partitioning in a pot experiment with Medicago truncatula in four contaminated soils. Soil Biol Biochem 40:2710–2712 Pawlowska TE, Charvat I (2004) Heavy-metal stress and developmental patterns of arbuscular mycorrhizal fungi. Appl Environ Microbiol 70(11):6643–6649 Rashid A, Ayub N, Ahmad T, Gul J, Khan AG (2009) Phytoaccumulation prospects of cadmium and zinc by mycorrhizal plant
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1986 species growing in industrially polluted soils. Environ Geochem Health 31(1):91–98 Shen H, Christie P, Li X (2006) Uptake of zinc, cadmium and phosphorus by arbuscular mycorrhizal maize (Zea mays L.) from a low available phosphorus calcareous soil spiked with zinc and cadmium. Environ Geochem Health 28:111–119 Tang M, Chen H, Huang JC (2009) AM fungi effects on the growth and physiology of Zea mays seedlings under diesel stress. Soil Biol Biochem 41:936–940 Tonin C, Wandenkoornhuyse P, Joner EJ, Straczk EJ, Leyval C (2001) Assessment of arbuscular mycorrhizal fungi diversity in the rhizosphere of Viola calaminearia and effect of these fungi on heavy metal uptake by clover. Mycorrhiza 10:161–168 Veresoglou SD, Menexes G, Rillig MC (2012) Do arbuscular mycorrhizal fungi affect the allometric partition of host plant biomass to shoots and roots? A meta-analysis of studies from 1990 to 2010. Mycorrhiza 22:227–235 Vogel-Mikus K, Pongrac P, Kump P, Necemer M, Regvar M (2006) Colonisation of a Zn, Cd and Pb hyperaccumulator Thlaspi praecox Wulfen with indigenous arbuscular mycorrhizal fungal mixture induces changes in heavy metal and nutrient uptake. Environ Pollut 139(2):362–371
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L. Liu et al. Wang FY, Lin XG, Yin R (2007a) Effect of arbuscular mycorrhizal fungal inoculation on heavy metal accumulation of maize grown in a naturally contaminated soil. Int J Phytoremediation 9(4):345–353 Wang FY, Lin XG, Yin R (2007b) Role of microbial inoculation and chitosan in phytoextraction of Cu, Zn, Pb and Cd by Elsholtzia splendens—a field case. Environ Pollut 147(1):248–255 Weissenhorn I, Leyval C (1995) Root colonization of maize by a Cdsensitive and a Cd-tolerance Glomus mosseae and cadmium uptake in sand culture. Plant Soil 175:233–238 Xu P, Christie P, Liu Y, Zhang J, Li X (2008) The arbuscular mycorrhizal fungus Glomus mosseae can enhance arsenic tolerance in Medicago truncatula by increasing plant phosphorus status and restricting arsenate uptake. Environ Pollut 156:215–220 Zarei M, Hempel S, Wubet T, Scha¨fer T, Savaghebi G, Jouzani GS, Nekouei MK, Buscot F (2010) Molecular diversity of arbuscular mycorrhizal fungi in relation to soil chemical properties and heavy metal contamination. Environ Pollut 158:2757–2765 Zhang XH, Zhu YG, Chen BD (2005) Arbuscular mycorrhizal fungi contribute to resistance of upland rice to combined metal contamination of soil. J Plant Nutr 28:2065–2077
Growth, cadmium uptake and accumulation of maize (Zea mays L.) under the effects of arbuscular mycorrhizal fungi Lingzhi Liu • Zongqiang Gong • Yulong Zhang Peijun Li
•
Accepted: 9 August 2014 / Published online: 5 September 2014 Ó Springer Science+Business Media New York 2014
Abstract The effects of three arbuscular mycorrhizal fungi isolates on Cd uptake and accumulation by maize (Zea mays L.) were investigated in a planted pot experiment. Plants were inoculated with Glomus intraradices, Glomus constrictum and Glomus mosseae at three different Cd concentrations. The results showed that root colonization increased with Cd addition during a 6-week growth period, however, the fungal density on roots decreased after 9-week growth in the treatments with G. constrictum and G. mosseae isolates. The percentage of mycorrhizal colonization by the three arbuscular mycorrhizal fungi isolates ranged from 22.7 to 72.3 %. Arbuscular mycorrhizal fungi inoculations decreased maize biomass especially during the first 6-week growth before Cd addition, and this inhibitory effect was less significant with Cd addition and growth time. Cd concentrations and uptake in maize plants increased with arbuscular mycorrhizal fungi colonization at low Cd concentration (0.02 mM): nonetheless, it decreased at high Cd concentration (0.20 mM) after 6-week growth period. Inoculation with G. constrictum isolates enhanced the root Cd concentrations and uptake, but G. mosseae isolates showed the opposite results at high Cd concentration level after 9 week growth period, as compared to non-mycorrhizal plants. In conclusion, maize plants inoculated with arbuscular mycorrhizal fungi were less sensitive to Cd stress than uninoculated plants. G.
L. Liu (&) Y. Zhang College of Land and Environment, Shenyang Agricultural University, Shenyang 110086, People’s Republic of China e-mail: [email protected] L. Liu Z. Gong P. Li Institute of Applied Ecology, Chinese Academy of Science, Shenyang 110016, People’s Republic of China
constrictum isolates enhanced Cd phytostabilization and G. mosseae isolates reduced Cd uptake in maize (Z. mays L.). Keywords Arbuscular mycorrhiza Zea mays L. Cd accumulation Phytoremediation
Introduction Cadmium (Cd) is a potentially phytotoxic heavy metal (HM). It is released into the environment by anthropogenic activities including mining, industry, and application of sewage sludge to agricultural land, on most occasions resulting in accumulation of Cd in soils (Janouskova´ et al. 2006). Soil pollution by Cd become a critical environmental concern due to its potential adverse ecological effects. High cadmium concentration in the soil has detrimental effects on ecosystem and is a risk to human health due to cadmium accumulation in food chains (Garg and Bhandari 2012; Glassman and Casper 2012). Cd is not biodegradable, remediation of Cd contaminated soil is challenging due to its unique properties. The use of physical or chemical methods for this purpose is often not cost effective, thus phytoremediation is becoming an increasingly important technology in soil remediation by offering an inexpensive and sustainable approach based on the immobilization of Cd within roots (phytostabilization) or to import and store Cd in the plant’s above-ground tissues (phytoextration) (Rashid et al. 2009). However, phytoremediation is a slow process and it is necessary to improve the efficiency to increase the stabilization or removal of Cd from soils. Arbuscular mycorrhizal fungi (AMF) provide an attractive system to advance phytostabilization of Cd by compounds secreted by the fungus, precipitation in polyphosphate granules in the soil,
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adsorption to fungal cell walls, and chelation of Cd inside the fungus (Glassman and Casper 2012; Jankong and Visoottiviseth 2008; Tang et al. 2009). Therefore, the use of mycorrhizal plants for land remediation has been proposed (Zarei et al. 2010; Carrasco et al. 2011; Go¨hre and Paszkowski 2006) with results such as mycorrhizae influencing metal transfer in plants by increasing plant biomass and plant phosphorus nutrition (Shen et al. 2006; Liu et al. 2011), reducing metal toxicity to plants by decreasing root to shoot HM translocation and shoot HM concentrations (Paul-Olivier et al. 2008). Many results showed that several factors such as AMF isolates, plant species, soil contamination levels and soil characteristics could determine the symbiotic conditions between the plants and the AMF isolates (Veresoglou et al. 2012). The effect of AMF on the uptake of Cd by plants is different with different reports. Among these researches, much attention has been paid to the influence of a single AMF isolate in the uptake and accumulation of metals by plants, while whether the similar AMF species would showed the same results is not yet totally clear. To use the arbuscular mycorrhiza (AM) symbiosis for phytoremediation, it is important to understand the compatibility between host plants and AMF isolates under the Cd contaminated conditions. Examination of the effect of Cd on symbiotic fungal life stages of two Glomus species showed Glomus intraradices to be more tolerant to Cd than Glomus etunicatum (Pawlowska and Charvat 2004). Zhang et al. (2005) reported that among the three tested Glomus species, Glomus mosseae inoculation gave the most protective effects on the upland rice under the combined soil contamination. These results showed that selection a compatible AMF isolate and plant may be useful for phytoremediation. Maize is a high biomass crops with increased ability to tolerate and accumulate Cd by introduction of metalbinding proteins or peptides and therefore is considered and tested as an alternative. However, studies of maize plants with different AMF isolates showed different results. Chen et al. (2004) and Liang et al. (2009) reported that inoculation with G. mosseae could enhance the biomass of maize plants and reduce the Cd translocation from root to shoot by immobilizating much Cd in roots. However, other studies showed that Glomus caledonium had no significant or negative effect on maize biomass and sent more Cd to shoots (Liao et al. 2003; Wang et al. 2007a, b), and Shen et al. (2006) showed that Cd addition level would influence the mycorrhizal effect. Therefore, the symbiotic effect of Glomus species and maize plant at different Cd addition level still needs to further study to meet with the phytoremediatione. The objective of this study is to investigate whether AM could decrease the Cd uptake and accumulation in maize
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plants under different Cd addition level. Three AMF, G. intraradices (GI), Glomus constrictum (GC) and G. mosseae (GM) were selected to colonize maize plants.
Materials and methods Plant, soil and AMF inoculum AM fungal inoculums used in the experiment consisted of G. intraradices (BGC USA05), G. constrictum (BGC USA02) and G. mosseae (BGC NM04A). The three AMF isolates were propagated in the laboratory in pot cultures during 4 months’ cycles with sorghum seedlings growing in pots containing a 1:1 (v/v) mixture of soil and sand. Inoculums containing rhizosphere soil, root, mycelium and spores of the three AMF isolates were applied to Zea mays growing in soils at different Cd levels, respectively. The soil used in the experiment was collected from Shenyang agriculture university, Liaoning Province, North China, with the following properties: pH (in H2O) 6.0, available K 20.26 mg kg-1, total C 2.83 %, total N 0.43 g kg-1, available P 14.84 mg kg-1. The soil was mixed with washed river sand (sand/soil, 1:2 v/v). Experimental design Seeds of Z. mays L.were surface sterilized with 10 % (v/v) hydrogen peroxide for 15 min, rinsed with sterile distilled water for 5–6 times. Sterilized seeds were germinated on Petri dishes lined with moisturized filter paper and placed in the dark at 25 °C for 2–3 days. Uniform seedlings were then selected and transferred to plastic containers (13 cm diameter, and 13 cm height) containing washed river sand (\2 mm) autoclaved at 121 °C for 1 h on two consecutive days. The experimental pots were disinfected with 70 % ethanol before soil was loaded. Each pot was first filled with 1.0 kg prepared mixture of soil and sand, then 50 g of inoculums was spread over the surface; for the nonmycorrhizae control the inoculums had been autoclaved. Finally the pots were covered with 0.25 kg prepared mixture of soil and sand. Four replicates were performed for individual treatments. The plants grew in a controlled environmental growth room, with temperatures of 25/18 °C (day/night), a photoperiod of 14 h at a quantum flux density of 400 lmol m-2 s-1 and 50 % relative humidity. The plants were watered with tap water as necessary and received 1/4-strength Hoagland modified solution, with a reduced P concentration (0.1 mM) once every 2 weeks. The pots were in a completely randomized design and were randomly rotated at weekly intervals to avoid site effects within the growth room.
Contribution of arbuscular mycorrhizal fungi in soil remediation
80
a
a
a
60
ab
a
b b
b
40 c
20
b Root colonization (%)
a Root colonization (%)
Fig. 1 Effects of cadmium addition on Zea mays L. root colonization by AMF (a 6 weeks, b 9 weeks). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
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80
a
a
a
a ab
60
b
b b
40
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20 0
No Cd
0.02 mM
0.2 mM
No Cd
0.02 mM
0.2 mM
Cd addition level GI
After the inoculation period, maize plants were to Cd addition. There were three AMF inoculum (GI, GC and GM inoculums) and four Cd levels (0, 0.02, 0.20 mM) with non-mycorrhizal control (NM). Forty milliliters of 1/4strength Hoagland modified solution with a reduced P concentration (0.1 mM) and with three different concentrations of Cd was added as CdCl2 weekly to each corresponding pots.
GC
GM
were performed using Duncan’s multiple range test (P \ 0.05). Prior to statistical analysis, data were logarithmically transformed [y = ln(x ? 1)] to satisfy the assumptions of the ANOVA. Software SPSS 11.5 was used for all the statistical analyses. Sigmaplot 10.0 for Cartography.
Results Harvest and analyses Mycorrhizal colonization Plants were harvested after 6- and 9-week, respectively. Shoots were first cut off, and roots were collected, washed gently with tap water, and then rinsed in deionized water. The clean roots were cut into segments about 1 cm long. A randomly selected subsample of one fresh root (0.2 mg) was taken for assessment of root colonization. Percentages of root colonization by the AMF were estimated by the gridline intersection method at 40–1609 magnification after clearing and staining the roots with trypan blue in lactoglycerol (Koske and Gemma 1989). Biomasses of the plant roots and shoots were determined after drying at 70 °C for 3 days. For the analysis of Cd concentrations in the root and shoot samples, the plant samples were ground, digested by HNO3 and HClO4 (3:1), and the Cd concentrations were determined by atomic absorption spectrometry with flameless atomization. Total Cd (accumulation) in the whole plant was calculated by the following equation: Total Cd = shoot Cd concentration (lg g-1) 9 shoot biomass (g) ? root Cd concentration (lg g-1) 9 root biomass (g). Statistical analysis Data were analyzed with a two-way analysis of variance (ANOVA) to assess the effects of AMF inoculation and cd levels in the experiment, for percentage of root length colonization, the NM control was excluded from the analysis due to no colonization. Comparisons between means
No root colonization was found in the NM control before the onset and after the treatment. Root colonization increased with increasing Cd addition except for a slight decrease for GC treatment at high Cd addition (Fig. 1a). Root colonization was significantly influenced by AMF inoculation (F = 39.21, P \ 0.001), Cd addition level (F = 35.7, P \ 0.001), and the interaction between AMF and Cd concentrations (F = 20.47, P \ 0.001). Cadmium addition (0.20 mM) inhibited root colonization in the GM and GC treatments (Fig. 1b). Generally, the percentage of colonization in mycorrhizal maize ranged from 30.3 to 72.3 % under different Cd concentrations. Among the three isolates, GI inoculation showed the highest root colonization as compared with GM and GC treatments. Plant biomass Cd decreased plant growth in the NM control treatment. Nonetheless, mycorrhizal colonization sustained the shoot and root biomass during the first 6-week growth period without Cd addition (Fig. 2a; Table 1). However, this inhibitory effect got less significant for shoot biomass after 9-week growth (Table 1) and GC and GM inoculation significantly increased the plant biomass (P \ 0.05) at 0.02 mM Cd addition level (Fig. 2b), compared with the NM control. Among the three AMF isolates, the biomasses of Z. mays L. did not differ significantly between
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a
10 8
b
a b
14
b
bc
bc
b
b bc
c
6 4 2
a
a
12
b b bc
Total biomass (g)
Fig. 2 Effects of cadmium addition and AMF inoculation on biomass of Zea mays L (a 6 weeks, b 9 weeks). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
Total biomass (g)
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b
10
a b
b
b
b
b
b
b
8 6 4 2
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0 0
0.02 mM
0.20 mM
0
0.02 mM
0.20 mM
Cd addition level GI
Table 1 Effect of different AMF on the growth of Zea mays L. under Cd stress
Cd addition level (mM)
Root to shoot ratio
6 week
9 week
6 week
9 week
6 week
9 week
GI
4.9b
6.42b
1.86cd
3.52 fg
0.38d
0.55def
GC
4.88b
6.32b
1.91cd
3.88de
0.39d
0.61bcd
GM
4.61bc
6.39b
1.81d
3.25 g
0.39d
0.51f
NM
5.88a
6.24b
2.78a
5.13a
0.47b
0.82a
GI
4.12c
6.40b
1.75d
3.72ef
0.43bcd
0.58cdef
GC
4.53bc
7.51a
1.87cd
3.95cde
0.42bcd
0.53ef
GM
4.84b
7.25a
2.03bc
4.30b
0.42cd
0.59cde
NM GI
4.61bc 4.57bc
6.16b 5.98b
2.14bc 1.80d
4.19bc 4.07bcd
0.47bc 0.39d
0.68b 0.69b
GC
4.78b
6.42b
1.90cd
3.41g
0.40 cd
0.53ef
GM
4.13c
6.38b
2.22b
3.80def
0.54a
0.60cde
NM
4.7bc
6.00b
2.05bc
3.83de
0.44bcd
0.64bc
Mycorrhiza
**
***
***
***
**
***
Cd level
***
***
n.s.
**
n.s.
n.s.
Mycorrhiza 9 Cd level
**
*
***
***
**
***
Different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
0.20
a
Significancea due to:
n.s. non-significant effect
treatments under the same Cd addition level, except for 0.02 mM of Cd addition at 9-week growth. Plant Cd concentrations Cd addition level and AMF inoculation significant influenced Cd concentrations in the shoots (P \ 0.01) and roots (P \ 0.01) of maize. After 6-week treatment period, maize with AMF inoculation had higher Cd concentrations in the shoot and root than that in the non-inoculated treatment at low Cd addition level (Fig. 3a, b). However, this effect became not significant or even a metal uptake decrease appeared on shoot biomass after 9-week growth at this Cd addition level. In the treatment with 0.2 mM Cd, mycorrhizal colonization decreased Cd concentration in maize slightly after 6-week growth compared to the non-inoculated treatment (Fig. 3c, d). However, effects of the three AMF isolates on Cd concentrations in the root of maize
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NM
Root biomass (g)
0.02
Significant effects according to two-way ANOVA: * P \ 0.05; ** P \ 0.01; *** P \ 0.001
GM
Shoot biomass (g)
0
Inoculation type
GC
were different after 9-week growth, GC and GM inoculation significantly enhanced and decreased Cd concentration (P \ 0.05), respectively, while GI inoculation showed no effect on the Cd concentration. Plant Cd uptake and accumulation At low Cd addition level, AMF inoculation enhanced total Cd uptake in the plants in the first harvest, however the AMF inoculation reduced Cd uptake in the final harvest. With increasing Cd addition level, AMF inoculation significantly decreased Cd uptake in the plants during 6-week growths. Inoculation with GC, GM and GI isolates significantly increased (P \ 0.05), decreased (P \ 0.05) and had no effect on the Cd uptake in the plants, compared with that in the non-inoculated plants (Fig. 4a, b). The ratio of Cd in the roots to the shoots showed a large differences in the three AMF inoculations as compared to
Contribution of arbuscular mycorrhizal fungi in soil remediation
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-1
Root concentration (mg kg )
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-1
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d
Root concentration (mg kg )
-1
Shoot concentration (mg kg )
a
Shoot concentration (mg kg )
Fig. 3 Effects of cadmium addition and AMF inoculation on Cd concentration of root and shoot of Zea mays L. (a, b 0.02 mM Cd, c, d 0.2 mM Cd). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
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Cd addition level
300
b
bc c
200 ab
b
a
c
100
0
9w
6w
9w
Harvest time GC
GI
a
250
b
NM
800 b
200
Total Cd uptake (ug)
a
Total Cd uptake (ug)
Fig. 4 Effects of cadmium addition and AMF inoculation on Cd accumulation in Zea mays L (a 6 weeks, b 9 weeks). Sample numbers (n) = 4, error bar = 1 standard deviation, different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05)
GM
b c c
150 a b
ab b
0 0.02mM
600
a
b
c
b
ab
a
c
0
0.20mM
0.02 mM
0.20 mM
Cd addition level GI
the NM treatments (P \ 0.05) at high Cd concentration in 9-week growths (Table 2). The higher root/shoot ratio indicated that plants with GC and GM inoculation Tran located less Cd from root to shoot than the NM treatment, and Cd uptake decreased by 83.1 and 68.0 %, respectively compared with the NM control. Although GM isolates increased Cd allocation to roots, the root Cd uptake in the plants was still 0.81 times to the NM control because of the low total Cd uptake in plants. To GI inoculation, it almost had no difference with NM treatment in Cd uptake and accumulation.
Discussion AMF colonization and plant biomass Studies have shown that high levels of heavy metals can inhibit, or even eliminate AMF colonization (Miransari
GC
GM
NM
2011; Weissenhorn and Leyval 1995). In contrast, some other studies found that the Cd contamination did not affect the root colonization (Chen et al. 2004; de Andrade et al. 2008). In our study, high levels of Cd concentrations had a strong effect on AMF development, and the three isolates exhibited different responses to the Cd, with GC and GM colonization being decreased significantly and GI being increased slightly in 9-week growths. The addition of Cd had no significant effect on colonization by GI isolates during the treatment period, showing that the association between GI isolates and Z. mays L. could be well established in Cd contaminated soils, GI inoculation had no effect on biomass enhancement, Cd uptake and allocation in maize plants, compare to NM treatment. The root colonization of 30–70 % observed in the pot experiment indicated that different level of compatibility between host plants and AMF isolates could occur under the Cd contaminated conditions. Growth time, Cd addition levels, and
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Table 2 Effect of different AMF isolates on the Cd uptake in shoots and roots of Zea mays L. under Cd stress Cd addition level (mM) 0.02
Inoculation type
Shoot Cd uptake (lg)
Root Cd uptake (lg)
Root to shoot Cd uptake ratio
6 week
6 week
6 week
9 week
9 week
9 week
GI
2.44d
3.44d
9.40d
12.37d
3.91b
3.62ab
GC
2.13d
8.27d
7.81d
14.12d
3.71b
1.71ef
GM
2.37d
7.65d
8.85d
16.01d
3.74b
2.10de
NM
1.93d
11.78d
8.11d
14.85d
4.21b
1.29f
GI
48.04b
190.50a
105.33b
465.99b
2.21c
2.49cde
GC
52.47a
157.46b
90.04c
529.17a
1.74c
3.41ab
GM NM
27.26c 56.13a
128.87c 189.47a
142.21a 135.10a
380.22c 470.60b
5.30a 2.42c
2.98bc 2.50cde
Mycorrhiza
***
***
***
**
***
n.s.
Cd level
***
***
***
***
***
***
Mycorrhiza 9 Cd level
***
***
**
***
***
***
0.20
Significancea due to:
Different letters above the columns indicate significant difference between means by Duncan’s Multiple Range Test (P \ 0.05) a
Significant effects according to two-way ANOVA: * P \ 0.05; ** P \ 0.01; *** P \ 0.001
n.s. non-significant effect
AMF isolates used might have affected the symbiotic status between the maize plants and the AMF isolates (Zhang et al. 2005). Previous studies showed that mycorrhizal plants had higher plant biomass compared to non-mycorrhizal plants (Leung et al. 2007, 2010; Xu et al. 2008). Differently, Liao et al. (2003) and Wang et al. (2007a, b) reported a decrease in plant biomass with Mycorrhizal colonization. Our study in the initial harvest was consistent with the latter. Although the inhibition from AMF disappeared gradually with the growth and Cd addition, and an obvious decrease in dry weight occurred in the non-inoculated plants with Cd addition, almost no positive effect of AMF on Z. mays L. resistance to Cd was observed. The growth responses to mycorrhiza might be due to differences in isolates of three AMF, plant species, soil characteristics used in the experiment. (Liao et al. 2003; Zhang et al. 2005). This lack of mycorrhizal effect on plant size is, however, an important requisite to demonstrate mycorrhizal influence on Cd uptake and accumulation without confounding dilution effects. Cd uptake and accumulation In our study, Z. mays L. colonized by AMF, especially at low Cd addition levels, had higher Cd concentrations in both shoots and roots in the first harvest, however, similar and lower Cd concentration in roots and shoots in the final harvest. With increasing Cd addition level, AM colonization reduced Cd concentration in the plants both during 6and 9-week growths. While GC isolates inoculation
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increased root Cd concentration in plants in 9-week growths. These results showed that AMF enhance Cd uptake in plants at low Cd addition levels and restrict Cd translocation from soil to plant at high Cd stress to reduce Cd toxicity to the plants, which is in accordance with the study by Vogel-Mikus et al. (2006), showing that inoculation with AMF decreased Zn and Cd concentrations of plant leaves at high soil metal concentration, and increased metal concentrations of plant leaves at low soil concentration. The effect of AMF on metal uptake in plants is different from other studies reporting that AMF increased uptake with reduced (Huang et al. 2006; Liang et al. 2009) or increased (Citterio et al. 2005) transfer to shoots and decreased uptake (Chen et al. 2003), sometimes even no uptake effects (Guo et al. 1996). Plants inoculated with three AMF isolates, especially in the presence of high Cd levels, had lower Cd uptake in the plants in the first harvest, however showed different results in the second harvest, GC, GM and GI isolates inoculation had higher, lower and the same Cd uptake in the plants than that in the nonmycorrhizal plants, respectively. Since mycorrhizal Z. mays L. did not have higher biomass than non-mycorrhizal Z. mays L. at high Cd addition level, the different results in three AMF isolates inoculation may attribute to different protection mechanism in the resistance of mycorrhizal plants to Cd stress. AM colonization immobilizes HM within the soil or within roots and therefore improves phytostabilization. Gaur (2004) and Gonza´lez et al. (2004) reported that extraradical hyphae from AMF can transport metals from soils to plants, but the transfer from fungus to
Contribution of arbuscular mycorrhizal fungi in soil remediation
plant can be restricted due to immobilization in the root tissues by compounds secreted by the fungus, precipitation in polyphosphate granules in the soil, adsorption to fungal cell walls and chelation of metals inside the fungus. In this study, the higher root to shoot Cd uptake ratio in GC and GM inoculation than NM plants at the high levels of Cd addition suggested that the proper AMF would immobilize Cd in their mycelium or induced metal-binding capacity in Z. mays L. roots, thus relatively less of the Cd could be translocated to the shoot (Tonin et al. 2001; Chen et al. 2004; Luis et al. 2006). With creating a more balanced environment in symbioses, AM colonization seems to improve the roots to cope with higher Cd concentrations ultimately. Among the three AMF isolates, GC isolates showed the potential use in Cd phytostabilization by absorbing more Cd in roots and GM isolates showed the potential in improving food quality by reducing Cd uptake in plants, especially in translocation to shoots. The results from above may be useful in appreciate mycorrhizal symbioses selection for different purposes. In general, the GC and GM isolates decreased the Cd translocation from the roots to shoot in the inoculated maize plants indicating the potential that some AMF may have in stress tolerance in Cd contaminated soils. Thus, by limiting Cd uptake and trapping this toxic element in the mycorrhizal symbiont, GM and GC isolates may contribute to reduce Cd entering the food chain and show a potential in phytostabilization applications. Acknowledgments Financial supports from NSFC (40930739 and 31101504) and Ministry of Environmental Protection of China, and arbuscular mycorrhizal isolates provided by Prof. Y. S. Wang (Beijing Academy of Agriculture and Forestry, China) are gratefully acknowledged. Conflict of interest of interest.
The authors declare that they have no conflict
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