A greenhouse experiment was conducted to investigate the role of arbuscular mycorrhizal fungi (AMF) on cadmium (Cd) and phosphorus (P) uptake by Sedum alfredii Hance (a Cd hyperaccumulator), and Ipomoea aquatica Forks from Cd-contaminated vegetable soils. In this experiment, four treatments were conducted: (1) I. aquatica (monoculture); (2) S. alfredii with I. aquatica (interculture); (3) interculture with GC fungi (GC: Glomus caledonium 90036); and (4) interculture with GV fungi (GV: Glomus versiforme HUN02B). The effects of AMF on colonization rate, plant biomass, Cd and P contents of day 56 S. Alfredii, the effects of S. alfredii and AMF on soil pH, soil EC, soil available Cd and P contents, soil total Cd content and soil acid phosphatase activity, the effects of S. alfredii and AMF on fresh biomass, Cd and P content of I. aquatica were examined. Comparing to control, it was found that AMF increased the colonization rate, plant biomass, Cd and P uptake of day 56 S. Alfredii, while S. alfredii and AMF reduced soil available Cd and P contents and increased soil acid phosphatase activity. S. alfredii did not affect the growth and production of I. aquatica. Furthermore, with AMF inoculation, I. aquatica P uptake was increased and Cd uptake was reduced. All these results indicated that inoculation of AMF and S. alfredii, there was a significant reduction (p<0.05) in Cd content in edible vegetables (I. aquatica), which in turn would improve the food safety.
Introduction
1.1 Cadmium pollution in soils
Cadmium (Cd) is a commonly known highly toxic metal. Among the top 20 toxins, Cd has been ranked number 7. It is mainly due to its negative influence on the enzymatic systems of cells (Al-Khedhairy et al., 2001; Sanità et al., 1999). Nowadays, in different countries, large areas of land have been contaminated by Cd and other heavy metals, due to the application of sludge or urban composts, fertilizers, pesticides, emissions from waste incinerators, waste water irrigation, residues from metalliferous mining, and the metal smelting industry (McGrath et al., 2001; Reeves and Baker, 2000; Yang et al., 2002). Furthermore, Cd is easily transferred to human food chain. It is no doubt that Cd contamination is a great threat to human health. Hence, these cause limiting of marketing agricultural products and reducing the profitability of the agricultural industry. Moreover, Cd can stay in soil over thousands years (Alloway, 1995).
1.2 Cd-contaminated soils in China
Nowadays, because of the economic in China develops rapidly, heavy metal contamination of agricultural soils has also become increasingly serious in China (Li et al., 1997a, b; Chen et al., 1999). Furthermore, agriculture in China has become increasingly rely on agrochemical usage. (Li et al., 1997a, b) When continuous application of agrochemicals, it will potentially exacerbate the accumulation of heavy metals in agricultural soils over time (Siamwalla, 1996; Chen et al., 1999). Therefore, Cd-contaminated soils are serious problem in China.
1.3 Cd hyperaccumulator - S. alfredii.
In order to deal with the Cd-contaminated soil, phytoremediation is a commonly used technique. By using green plants, which offers the benefits of low cost, and environmentally sustainable, to clean up contaminated soils (Salt et al., 1998; Long et al., 2002). Yang, et al. (2004) proved that Sedum alfredii is a new powerful Cd hyperaccumulator, so it is a useful plant material for phytoremediation of Cd-contaminated soils and cause reduction of soil available Cd. In addition, it has characteristics of fast growth, large biomass, asexual reproduction, and perennial. Moreover, according to Yang et al. (2001), S. alfredii can grow up to 40 cm height and propagate 3-4 times in a year if the environmental conditions are favorable. Therefore, in my study, intercropping of S. alfredii and I. aquatica is choosen. S. alfredii were planted in order to reduce soil available Cd and in turn reduce the Cd uptake by I. aquatica.
1.4 Function of Arbuscular mycorrhizal fungal (AMF).
Arbuscular mycorrhizal fungal (AMF) symbioses are mutualistic interactions between fungi and most plants. So, plants obtain nutrients, particularly P, through symbiosis (Smith et al., 2003). AMF associations with plant roots, through the hyphae which explore the soil away from the root surface, so it increases the effective absorbing zone of the root (Wang et al., 2007). Nevertheless, some studies proved that AMF isolated from contaminated soils are able to tolerate higher metal concentrations (Gonzalez-Chavez et al., 2002; Vivas et al., 2005). Therefore, in my study, AMF were inoculated to enhance the phytoremediation power of S. alfredii.
1.5 Intercropping of S. alfredii and I. aquatica.
Ipomoea aquatica is a popular and common leafy vegetable in China. However, past research showed that I. aquatica can be easily contaminated by Cd in the soil (Wang, J. L et al. 2007). Therefore, in my study, S. alfredii Hance was used to phytoremediate the Cd-contaminated soil by uptaking more Cd from soil and reducing soil Cd content, in turn reduce Cd uptake by I. aquatica.
1.6 Experimental objectives
The objectives of this experiment were to investigate the effects of AMF on colonization rate, plant biomass, Cd and P content of Sedum alfredii. And, examine the effects of S. alfredii and AMF on soil pH, EC, total Cd content, bioavailable Cd content and P content and acid phosphatase activity of soil. Furthermore, to investigate the effects of Sedum alfredii and AMF on fresh biomass, Cd and P uptake of Ipomoea aquatica. The aim of my study was to enhance Cd uptake by S. alfredii and so reduce Cd content in I. aquatica. Therefore, reduce the risk of soil contaminants entering the human food chain.
Materials and Method
2.1 AM Fungal inocula
Two AMF inocula were used in this experiment. One contained only a AMF strain, Glomus caledonium 90036(GC), which was isolated from a fluvo-aquic soil in Hennan Province, China, and was deposited at the Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China. The other also contained only one AMF strain, Glomus versiforme HUN02B(GV), which was isolated from a grass soil in Hunnan Province, China, and was deposited at the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry. As a mixture of rhizospheric soil containing spores, hyphae, and mycorrhizal root fragments, the AM inocula were propagated on sudangrass [Sorghum sudanese (Piper) Stapf.] grown in an autoclaved (121°C for 1 h on three successive days) sandy soil in pots for two successive propagation cycles (two month each). At the same time, the non-mycorrhizal inoculum was also prepared with the same sterilized substratum on which sudangrass was cultivated under the same conditions. All inocula were air-dried and sieved (2 mm) before inoculation.
　2.2 Soil Preparation
The soils were collected from Guangzhou Viaoying village, which is a vegetable field. The dominant crop species planted on these site were tomato, beans, etc. As those harvested vegetable will be sell to Hong Kong, so we choose this field soil as our experiment samples. In this site, the soils were suspected being polluted. The possible pollution sources were river sediment, fertilizer, pesticides, etc. In the field area, 4 to 6 points were randomly choosen to collect the soil samples. Soil samples were collected 0-15 cm in depth, air-dried and sieved to < 2mm.
2.3 Experimental design
In this pot experiment, four treatments were conducted: (1) I. aquatica (monoculture); (2) S. alfredii with I. aquatica (interculture); (3) interculture with GC fungi (GC: Glomus caledonium 90036); and (4) interculture with GV fungi (GV: Glomus versiforme HUN02B). The Seeds of I. aquatica were brought from Hong Kong, the seeds were cv. Hantong. Pot were arranged in a randomized complete block design with five replicates per treatment.
The pot used were 24cm (radius) x 22cm (height). 2.4 kg of soil are based. Then, base soil were watered until water holding capacity reach 75%. On the top of the base soils, 150g of AMF were added. Seeds of I. aquatica were surface-sterilized with 0.5% NaCLO and subsequently washed several times with distilled water and germinated at 28 ℃ (12h) before sowing. 9 seeds were transplanted into each pot (Fig. 1) and after emergence 6 seedlings were left. Afterward, addition of 600g top soils without watering. After emergence, soil were watered until water holding capacity reach 50%. Plants were watered to maintain soil moisture at about 50% of water holding capacity by adding tap water suring the ecperimental period.
S. alfredii were harvested after 56 days of growth. Mycorrhizal colonization rate, dry biomass, Cd content and P content of day 56 S. alfredii were determined. After 28 days, totally 3 samples of I. aquatica were harvested in each pot. After 56 days, all the remaning plant samples were harvested. Fresh biomass, Cd content and P content of shoots and roots of day 28 and 56 I. aquatica were determined. Moreover, after 56 days, all soils are collected in order to determine soil pH, EC, total Cd content, bioavailable Cd content and P content and acid phosphatase activity.
Fig. 1. Figure shows the location of plantation and sowing in pot.
2.4 Mycorrhizal colonization rate
Root of day 56 S. alfredii were cleaned in 10% KOH, stained with 0.05% trypan blue (Philips and Hayman 1970). The clean roots were cut into segments of about 1 cm in length. A randomly selected fresh 100 segements of root samples were taken for the assessment of root colonization. The percent colonization was then determined by the grid intersect method (Giovannetti and Mosse 1980).
2.5 Chemical analyses
Soil samples were air-dried, ground, sieved through a 2mm screen, and then used to analyze the diethtylenetetraminepentaacetic acid (DTPA) extractable heavy metal concentrations (Lindsay and Norvell, 1978), pH and EC (electro-conductivity) and total Cd. The values of soil pH and electrical conductivity (EC) (soil:deionized water = 1:5) were measured by a pH meter (Beckman) and an EC meter (Orion 160), respectively. Soil samples were air-dried, ground, sieved through a 0.15mm screen were used to analyze total Cd. Soil sub-samples weighed 0.5g after oven drying at 105℃ for 12h. The soil sub-samples (0.5g) were mirowave digested by "CEM Corporation" Model Mars Xpress Microwave Digestion Systerm with concentrated HCL : HNO3 : HClO4 at 6:2:4 volume ratio. The total Cd concentration were then determined by Atomic Absorption Spectrometer (Spectr AA-20 Varian). A standard reference material: NIST SRM 2711a Montana Soil II was used to verify the accuracy of Cd determined in soils and the recovery rate was 94%. Soil available P was tested by the Bray and Kurtz (1945) method, which employs an acid extracting solution (0.025 M hydrochloric acid and 0.03 M ammonium fluoride), and the P concentration in the extracts was then determined using a UV-Vis spectrophotometer (UV-1061, Shimadzu, Kyoto) based on the molybdenum blue reaction (Lu, 2000). Soil samples were air-dried for the assay of soil acid phosphatase activity. It was determined by incubation at 37 °C with acetate buffer (pH 5) according to the method of Tabatabai (1982), and is given in units of mg p-nitrophenol produced g-1 soil 24 h-1.
Shoots and roots of I. aquatica samples were rinsed with tap water, then with deionized water, and weighed after oven drying at 70℃ for 48h. Then, plants samples were ground with a stainless steel grinder and passed through a 0.15mm sieve. For dried day 28 shoots and roots of I. aquatica, sub-samples were weighed 0.75g and 0.1 g respectively. For dried day 56 shoots and roots of I. aquatica, sub-samples were weighed 1g and 0.5g respectively. For dried day 56 shoots of S. alfredii, sub-samples were weighed 0.2g. They were digested by concentrated 8ml HNO3, were used to analyze total Cd and P content. The concentration of Cd was determined by Atomic Absorption Spectrometer (Spectr AA-20 Varian). A standard reference material: NIST SRM 1573a Tomato Leave was used to verify the accuracy of Cd determined in soils and the recovery rate was 96%. Concentrations of P were determined using molybdenum blue method (Page et al., 1982).
2.6 Statistical Analysis
The data were subjected to one-way analysis of variance (ANOVA) using the SPSS software program (SPSS Inc., Chicago, Version 10.0 for Windows) (Wang, Zheng, and Chao, 2003). Means and standard deviation were calculated for 5 replicate values. Means were compared by the Duncan's multiple range test (p < 0.05).
Results
3.1 Colonization rate, plant biomass, Cd content and P content of day 56 S. alfredii.
Although all S. alfredii were colonized (Table 1), there were significant difference (p < 0.05) between treatments. With inoculation of AMF, there was significant increase (p< 0.05) of mycorrhizal colonization in of day 56 S. alfredii. Furthremore, with inoculation of GC fungi, there was significant increase (p< 0.05) of day 56 S. alfredii dry biomass (Fig. 2). This indicated that with AMF colonization, the growth of S. alfredii is enhanced. On the other hand, with inoculation of AMF, there was significant increase (p < 0.05) of Cd content in day 56 S. alfredii (Fig 3A). This result indicated that S. alfredii has an extraordinary ability to absorb Cd and concentrate it in the aboveground tissue. However, there were no significant difference of P content among three treatments (Fig. 3B). Comparing two different AMF species, S. alfredii inoculated with GC fungi had significantly higher (p < 0.05) dry biomass and Cd content.
Table 1. Effects of AMF on colonization rate of day 56 S. alfredii in response to different treatment.
　
Average Mycorrhizal colonization (%)
Inter
18.0 (3.81) b
Inter + GC
42.4 (7.09) a
Inter + GV
35.4 (7.60) a
Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi. Mean values are presented with standard deviation in parentheses. Different letters in the same column show significant difference by Duncan's multiple range test at p < 0.05.
Fig. 2- Individual dry biomass (g) of day 56 Sedum alfredii with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05). (Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
(B)
Fig. 3- (A) Plant Cd content (mg kg-1 dry biomass) and (B) P content (g kg-1 dry biomass) of day 56 Sedum alfredii with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05). (Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
3.2 Day 56 soil pH, EC, total Cd content, bioavailable Cd content and P content and acid phosphatase activity.
In different treatments, all soil pH < 5.5, that means they are acidic (Fig. 4A). However, with intercropping, there was significant increase (p < 0.05) of soil pH. Moreover, with inoculation of AMF, there were significant increase (p < 0.05) of soil pH. Furthermore, with inoculation of GC fungi, it showed a higher ability to cause obvious increase (p < 0.05) of soil pH. On the other hand, there were no obvious difference of EC among treatments (Fig. 4B).
Although there were no significant difference of soil total Cd content among treatments (Fig 5A), there were significant decrease (p < 0.05) of soil available Cd content (Fig. 5B) in interculture with AMF inoculation. This indicates that intercropping with AMF inoculation, less Cd is available in soil, that means plants could uptake less Cd from soils.
Intercorpping caused reduction of soil bioavailable P and significant decrease (p < 0.05) of soil acid phosphatase activity (Fig. 6). Moreover, with inoculation of AMF, there were significant decrease (p < 0.05) of soil bioavailable P and significant increase (p < 0.05) of soil acid phosphatase activity (Fig. 6).
(B)
Fig. 4- (A) Soil pH and (B) EC (µS cm-1) of day 56 soils with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05). (Mono: monoculture of Ipomoea aquatica; Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
(B)
Fig. 5- (A) Total Cd content (mg kg-1) and (B) Soil bioavailable Cd (mg kg-1) of day 56 soils with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05(Mono: monoculture of Ipomoea aquatica; Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
(B)
Fig. 6- (A) Soil available P (mg kg-1) and (B) Soil acid phosphatase activity (mg g-1 24h-1) of day 56 soils with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05). (Mono: monoculture of Ipomoea aquatica; Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
3.3 Fresh biomass, Cd and P content of day 28 and 56 I. aquatica.
Comparing differment treatments of day 28 and 56 I. aquatica, day 56 I. aquatica had a higher fresh biomass, which are 3 times greater than that of day 28 I. aquatica (Fig. 7). There were no obvious difference of both day 28 and 56 I. aquatica shoot and root frsh biomass, except there was a significant increase (p < 0.05) of day 56 I. aquatica shoot fresh biomass in which intercropping with inoculation of GV fungi. Moreover, this result indicated that intercorpping did not affect the growth of I. aquatica.
Comparing monoculture and interculture, there were significant decrease (p < 0.05) of shoot Cd content in day 28 I. aquatica but no significant different in day 56 I. aquatica (Fig. 8). On the other hand, there were significant decrease ( p < 0.05) of root Cd content in both day 28 & 56 I. aquatica (Fig. 8). This indicated that intercropping is useful for reduce Cd uptake by I. aquatica. On the other hand, comparing interculture and interculture with the inoculation of AMF, there was no significant difference in day 28 I. aquatica shoot Cd content. However, in day 56 I. aquatica, there was significant reduction (p < 0.05) of shoot Cd content. Furthermore, with GC fungi inoculation, there were significant reduction (p < 0.05) of root Cd content in both day 28 and 56 I. aquatica (Fig. 8). These showed that AMF is useful to reduce the Cd uptake by I. aquatica .
In both day 28 and 56 I. aquatica, there were no obvious difference of shoot P content in all treatments (Fig. 9). Comparing monoculture and interculture, there were no significant difference of root P content in both Day 28 and 56 I. aquatica (Fig. 9). This indicated that intercropping did not affect P uptake by I. aquatica. Comparing interculture and interculture with GC fungi, there were significant increase (p < 0.05) of root P content in both day 28 and 56 I. aquatica. This indicated that intercroppping with GC fungi inoculation could enhance the P uptake by I. aquatica. On the other hand, with GV fungi inoculation, there was significant decrease (p < 0.05) of root P content in day 28 I. aquatica and there was no significant difference of root P content in day 56 I. aquatica.
(B)
Fig. 7- Shoot and root fresh biomass (g) of (A) day 28 and (B) day 56 Ipomoea aquatica with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05). (Mono: monoculture of Ipomoea aquatica; Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
(B)
Fig. 8- Shoot and root Cd content (mg kg-1 fresh biomass) of (A) day 28 and (B) day 56 Ipomoea aquatica with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05). (Mono: monoculture of Ipomoea aquatica; Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
(B)
Fig. 9- Shoot and root P content (g kg-1 dry biomass) of (A) day 28 and (B) day 56 Ipomoea aquatica with different treatments. All the data are means of 5 replications, and the bars represent standard deviation. In different treatment, means with different letters are significantly different (p< 0.05). (Mono: monoculture of Ipomoea aquatica; Inter: interculture of Sedum alfredii and Ipomoea aquatica; Inter+GC: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GC fungi; Inter+GV: interculture of Sedum alfredii and Ipomoea aquatica with inoculation of GV fungi.)
Discussion
The aim of this study was to examine the effect of AM fungi on Cd uptake by Sedum alfredii and the effect of AMF and Sedum alfredii on Cd uptake by Ipomoea aquatica from Cd-contaminated vegetable soils, so as to reduce Cd uptake by I. aquatica. It is known that when there are excessive concentrations of heavy metals in soils, it is not only toxic to plants, but also bacteria and fungi (Vivas et al., 2003). Moreover, Cadmium is highly toxic to plant. Therefore, in this study, intercropping with the addition of AMF was used in order to enhance S. alfredii uptake more Cd, so as to reduce soil available Cd and in turn reduce Cd uptake by I. aquatica.
4.1 Effects of arbuscular mycorrhizal fungi (AMF) on colonization rate, plant biomass, Cd content and P content of day 56 S. alfredii.
Although all S. alfredii were colonized by AMF (Table 1), there were significant difference (p < 0.05) between treatments. With inoculation of AMF, there was significant increased (p< 0.05) of mycorrhizal colonization in of day 56 S. alfredii. This result is in agreement with a few reports that AMF has ability to colonize hyperaccumulator plants and consequently increase plants biomass (Liu et al.,2004). Moreover, the result shows that inoculation of GC fungi increased soil pH better than that of GV fungi. This increased pH may account for the significant increased (p< 0.05) of day 56 S. alfredii biomass (Fig. 2). As the soil is less acidic, which is much more suitable for plants growth. Therefore, dry biomass of day 56 S. alfredii in interculture with inoculation of GC fungi was larger than that of interculture. This increase of dry biomass of day 56 S. alfredii together with the significant increase (p < 0.05) of Cd content in it (Fig. 3A) , and hence, increased Cd uptake. Furthermore, precious report found that some mycorrhizas may enhance the ability of plants to scavenge for limited and immobile nutrients, especially P (Smith et al., 2003), so the plant biomass incrased. This also account for that, with inoculation of GC fungi, there was significant increased (p< 0.05) of day 56 S. alfredii dry biomass (Fig. 2). Nevertheless, this reult is consistent with Shen, et al. (2006) reported that the AMF may facilitate plant metal tolerance in a few ways. For example, the mycorrhiza increased plant biomass, so as to dilute the metals content in the plant tissues, and indirectly by enhancing plant P nutrition with a possible further contribution to enhance plant biomass.
Although there were no obvious difference of P content among all treatments (Fig. 3B), there was significant increased (p< 0.05) of day 56 S. alfredii dry biomass in intercropping with GC fungi (Fig. 2), that means S. alfredii P uptake was increased. This result can be accounted by Smith and Read (1997), when AM hyphae absorb P, then translocate it rapidly and deliver P to the root, which is believed as the main basis for AMF positive effects on P uptake and plant growth. On the other hand, some researchers found that AMF can provide the governing route for plant P supply, even when overall plant growth or P uptake remains unchanged (Smith, Smith, and Jakobsen, 2003). This can be accounted for intercropping with GV fungi, although there were no signifcant differnce for dry biomass and P content of day 56 S. alfredii.
With inoculation of AMF, there were significant increased (p < 0.05) of Cd content in day 56 S. alfredii (Fig. 3A).This result is consistent with Yang, et al. (2004) reported that S. alfredii has an remarkable ability to absorb Cd and concentrate it in the aboveground tissues, particularly in the leaves. Moreover, Yang, et al. (2004) also proved that , shoot growth of S. alfredii can be enhanced slightly by Cd supply at suitable levels.This proves that S. alfredii has an extraordinary ability to hyperaccumulate Cd in its shoots, that means it is a suitable plant material for Cd phytoextraction in Cd-contaminated soils. However, the mechanisms of Cd uptake, transport and accumulation in hyperaccumulator plants are not fully known yet. It is suggested by Welch and Norvell (1999) that, it is believed that non-accumulator plants uptake Cd by the same carrier as for other divalent cations, for example, Zn2+, Fe2+, or Cu2+, or via cation, Ca2+ and Mg2+channels. The mechanisms behind the tolerance of S. alfredii to Cd needed further investigation.
To sum up, it is no doubt that there was an enhancement of S. alfredii Cd uptake by AMF. S. alfredii uptake more Cd and reduce soil available Cd. As a result, intercropping is useful to reduce Cd uptake by I. aquatica.
4.2 Effects of S. alfredii and AMF on soil pH, EC, total Cd content, bioavailable Cd content and P content and acid phosphatase activity.
Although in all treatments, soil pH < 5.5, that means they are acidic (Fig. 4A). However, with intercropping, there was significant increase (p < 0.05) of soil pH. As reported by Shen, et al. (2006), more acidic soil conditions may increase the severity of Cd toxicity. That means intercroping is useful for increase soil pH so as to reduce Cd toxicity to plant. On the other hand, there were no significant reduction of soil total Cd and soil available Cd (Fig. 5A and 5B) among different treatments, because of the short time period of experiment. However, it is proved by Yang, et al. (2004) that Sedum alfredii is a powerful Cd hyperaccumulator, so it is useful for phytoremediation of Cd-contaminated soils and hence could reduce soil available Cd. In addition, intercorpping caused reduction of soil bioavailable P and significant decrease (p < 0.05) of soil acid phosphatase activity (Fig. 6A and 6B). This is because both plants (S. alfredii and I. aquatica) absorbed nutrient from soil at the same time, in turn caused reduction of soil bioavailable P. Furthermore, Moreno et al. (2003) reported that as soil acid phosphatase was sensitive to heavy metals and could be used as toxicity test to establish the impact of heavy metals on the functioning of the soil. That means with intercropping, the function of soil is weakened.
According to the result, there were no obvious difference of EC among treatments (Fig. 4B). EC could be used to measure the dissolved salt or ion concentrartion in the soil, in which for crop absorption. It is known that if EC is too high, which is not good for crop growth. That means in different treatments, the soils condition are similar, which did not affect the experiment results interpretation.
With AMF inoculation, soil pH were increased significantly (p < 0.05). Furthermore, with inoculation of GC fungi, it showed a higher ability to cause obvious increase (p < 0.05) of soil pH (Fig. 4A). It is reported that AMF increased soil pH and causing metal less available for plant uptake (Shen, et al., 2006.). However, according to my result, AMF increased soil pH and making metal more available for hyperaccumulator uptake. On the other hand, although there were no significant difference of soil total Cd content among treatments (Fig 5A), there were significant decrease (p < 0.05) of soil available Cd content (Fig. 5B) in interculture with AMF inoculation. This showed that less Cd is available in soil, due to the enhancement of Cd uptake by hyperaccumulator and that means I. aquatica could uptake less Cd from soil. Moreover, with inoculation of AMF, there were significant decrease (p < 0.05) of soil bioavailable P (Fig. 6A). Because AMF enhance S. alfredii uptake more P, when I. aquatica uptake P at the same time, soil available P decreased. In addition, with inoculation of AMF, there were significant increase (p < 0.05) of soil acid phosphatase activity (Fig. 6B). This result was consistent with Vivas et al. (2005) that AM fungi can increase Cd-contaminated soil phosphatase activities. Also, in the P nutrition of plants, soil phosphatase may play an important role because it mediates the release of inorganic phosphorus from organically bound phosphorus (Wang, et al. 2007). Wang, et al. (2007) also suggested that soil enzymes are useful to indicate the AMF potential beneficial effects on soil quality. When there was significant increase (p < 0.05) of soil acid phosphatase activity, that means more P is available in soils for plants direct absoption. According to a few past studies, soil enzymatic activities could be used to indicate soil fertility (Stefanic et al., 1984), soil productivity (Busto and Perez-Mateos, 1997) and soil quality (Dick, 1994). The increasesd phosphatase activities by AMF inoculation indicate that AMF play an important role in Cd-contaminated soils improvement. Varma (1998) suggested that the mechanisms on the soil enzymatic activities enhancement may involve the role of AM fungi, as AM propagules themselves to synthesize soil enzymes. It is reported that AM fungal hyphae can produce some hydrolytic enzymes. Therefore, soil enzymatic activities is enhanced.
4.3 Effects of S. alfredii and AMF on fresh biomass, Cd and P content of day 28 and 56 I. aquatica.
When comparing monoculture and interculture, there were no significant reduction of both day 28 and 56 I. aquatica fresh biomass and P content (Fig. 7 and 9). These indicated that intercorpping did not affect the growth of I. aquatic. Moreover, there were significant decrease (p < 0.05) of shoot Cd content in day 28 I. aquatica but no obvious difference shown in day 56 I. aquatica (Fig. 8A and 8B). On the other hand, there were significant decrease (p < 0.05) of root Cd content in both day 28 and 56 I. aquatica (Fig. 8A and 8B). This is because with intercropping, I. aquatica could uptake less Cd as hyperaccumulator uptake more Cd from soil. This indicated that intercropping is useful for reduction of Cd uptake by I. aquatica. These results implied that intercropping is applicable, so that farmers could phytoremediate the contaminated soils with crops production at the same time. Onsite field studies are needed to further investigate its feasibility in farmland.
With GC fungi inoculation, there were no significant increase of day 28 and 56 I. aquatica fresh biomass (Fig. 7A and 7B), but there were significant reduction (p < 0.05) of root Cd content in both day 28 and 56 I. aquatic and of shoot Cd in day 56 I. aquatic (Fig. 8A and 8B). This is because as GC fungi significantly enhanced (p < 0.05) soil acid phosphatase activity (Fig. 6B), in turn increased P uptake by S. alfredii together with the significant enhancement (p < 0.05) of day 56 S. alfredii dry biomass (Fig. 2). Consequently, this significantly enhanced (p < 0.05) hyperaccumulator uptake more Cd (Fig. 3A), and caused a significantly decrease (p < 0.05) of soil bioavailable Cd (Fig. 6B). As a result, day 56 I. aquatica could uptake less Cd. Furthermore, there were significant increase (p < 0.05) of root P content in both day 28 and 56 I. aquatic (Fig. 9A and 9B). As suggested by a few similar studies, this reduced Cd uptake and increased P uptake may be because of Cd, which may be a phosphate analog and so it competes with P uptake because they are both taken up via the same phosphate transport systems (Ullrich-Eberius et al., 1989). However, further investigation is needed. To sum up, all these results proved that interculture with GC fungi inoculation is useful to enhance Cd uptake by S. alfredii and reduce Cd uptake by I. aquatica.
With GV fungi, there were no significant differences of root Cd content in both dy 28 & 56 I. aquatica (Fig. 8A and 8B). However, in day 56 I. aquatica, there was significant reduction (p < 0.05) of shoot Cd content (Fig. 8B). At the same time, there was significant increase (p < 0.05) of shoot fresh biomass in day 56 I. aquatica (Fig. 7B). Therefore, the Cd uptake by shoot of day 56 I. aquatica was reduced. In addition, as GV fungi enhanced Cd uptake by S. alfredii (Fig. 3A), causing a significant decrease (p < 0.05) of soil available Cd (Fig. 5B), so less Cd from soils could be uptake by I. aquatica. Moreover, this may also related to the P uptake by day 56 I. aquatica. There was an obvious increased (p < 0.05) of fresh biomass in shoot of day 56 I. aquatica (Fig. 7B), as its biomass increased, that means its P uptake would be increased, although there was no significant difference shown in P content of 56 I. aquatica (Fig. 9B). To sum up, all these results showed that interculture with GV fungi inoculation is useful to increase P uptake and reduce Cd uptake of I. aquatica. This may be due to the uptake competition between Cd and P, as they are both taken up via the phosphate transport systems (Meharg and Hartley-Whitaker, 2002).
Conclusion
The present study examined the effect of AMF on Cd and P uptake by S. alfredii and I. aquatica from cadmium-contaminated vegetable soils. S. alfredii is useful for phytoremediation and by using intercropping, the addition of hyperaccumulator did not affect other plants growth. However, further on site investigation is needed for the feasibility of intercropping in farmland. Moreover, intercropping with AMF inoculation is useful to significantly enhance (p < 0.05) Cd uptake by S. alfredii, consequently reduce soil available Cd. As a result, there was significantly reduction (p < 0.05) of Cd uptake by I. aquatic. This improves the food safety and sub-sequently reduces human Cd intake. Although Cd content in I. aquatica is reduced, it still cannot meet the State Environmental Protection Administration of China (1995) standard of Cd content < 0.05 mg/kg. Therefore, further investigations are needed to further reduction of Cd content in I. aquatica and in turn improve food safety.
