Cadmium and lead accumulation and low-molecular-weight organic acids secreted by roots in an intercropping of a cadmium accumulator Sonchus asper L. with Vicia faba L.

Abstract

Sonchus asper L. and Vicia faba L. are a local cadmium (Cd) accumulator and a main winter crop, respectively, found in the Huize lead–zinc mining area in Yunnan Province, Southwest China. The biomass and low-molecular-weight organic acids (LMWOAs) secreted by the roots of these plants, Cd and lead (Pb) contents and their accumulation in a S. asper monoculture, V. faba monoculture and S. asper/V. faba intercrop were investigated in a field experiment at 35, 80 and 180 d after planting. The results showed that (1) intercropping had no notable influences on plant biomass and grain yields of V. faba but led to a significant increase in the amount of stem and leaf biomass of S. asper at 180 d after planting. (2) The major LMWOAs secreted by the roots of both V. faba and S. asper were oxalic acid, tartaric acid and citric acid. Intercropping resulted in an increase and decrease in the LMWOA contents secreted by V. faba and S. asper roots, respectively. (3) Along with plant growth, the available Cd content decreased and the available Pb contents did not exhibit obvious changes in the soil samples of a V. faba monoculture. The amount of available Cd and Pb both increased in the soil of the S. asper monoculture, but decreased in that of the S. asper/V. faba intercrop. (4) Intercropping resulted in a decrease in the contents and accumulation of Cd and Pb in V. faba plants, but an increase in both the contents and accumulation of Cd and Pb in S. asper plants. Moreover, intercropping enhanced the enrichment and translation coefficients of Cd for S. asper. The remediation efficiency was the highest at 180 d after planting. (5) There were significant negative correlations between the contents of citric acid, malic acid (secreted by V. faba roots), oxalic acid and tartaric acid (secreted by S. asper roots) and the available Cd content in the soil samples. In addition, there was a significant positive correlation between the available Cd content in the soil and the Cd contents in the roots and grains of V. faba. Intercropping reduced the Cd contents in the plants and grains of V. faba and was closely related to the decrease in the available Cd content in the soil samples, which was mediated by plant roots that secreted LMWOAs.

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1. Introduction

Heavy metal pollution in farmlands is a major environmental problem and is a cause for global concern. Production activities, such as waste-water irrigation, sludge for agricultural utilization, the exploitation and smelting of mineral resources and pile-up of harmful waste, result in a diffusion of heavy metals, such as cadmium (Cd), lead (Pb), zinc (Zn) and arsenic (As), into farmland soils.¹ The heavy metals released into farmlands are absorbed by crops, subsequently hindering the growth of crops and increasing the heavy metals contents of the edible parts of the crops. Together, heavy metals seriously threaten food safety and human health through the food chain.^2,3 Therefore, the remediation of heavy metal-polluted farmlands and reduction of the heavy metal contents in agricultural products grown in polluted farmlands have drawn much attention.

Among the remediation technologies available for treating heavy metal-polluted soils, phytoremediation has several advantages such as ease of implementation in the field, relatively low cost, soil improvement and lack of secondary pollution; therefore, it has become one of the major methods for the remediation of heavy metal-polluted farmlands.⁴ However, it also has some deficiencies, such as slow growth of the accumulators, small biomass, and restriction by climatic factors, which makes phytoremediation a long-term and low-efficiency process. These deficiencies have limited the wider application of phytoremediation in heavy metal-polluted farmlands.⁵ In addition, phytoremediation requires agricultural production to stop, which does not conform to the national conditions of China (i.e., “an enormous population but with less per capita cultivated land”); this makes popularization and application of this technology difficult.⁶

Intercropping is a significant agricultural planting measure that not only promotes the effective use of agricultural resources, such as soil nutrients, water and light, but also has prominent ecological and environmental benefits.^7,8 The intercropping of an accumulator (or hyperaccumulator) with a crop was used to remediate heavy metal-polluted soils and was found to have many advantages. Intercropping allows people to carry out agricultural activities and to apply accumulators or hyperaccumulators to remediate polluted soil at the same time. Some intercropping patterns of accumulators with crops have been applied to heavy metal-polluted soils such as Solanum nigrum/onion,⁹ Sedum alfredii/maize,¹⁰ S. alfredii/upland kangkong,¹¹ Brassica juncea/alfalfa,¹² and Pteris vittata/Panax notoginseng intercropping.¹³ These intercropping patterns do not require the suspension of agricultural production, whereas the accumulator is used to remediate the heavy metal-polluted soils, reduce the heavy metal contents in crop products to meet safety and quality requirements and to execute “agricultural production accompanied by remediation”. Therefore, this method exerts a positive influence on both the remediation of polluted farmlands and the safety of agricultural products.^14,15 People have used this method to simultaneously facilitate remediation and agricultural production, and it is considered to be more suitable for polluted farmlands in China.^16,17 However, the mechanisms of how intercropping influences heavy metal accumulation in the accumulators and crops remain unclear.

Under heavy metals stress, the secretion of low-molecular-weight organic acids (LMWOAs) by plant roots is commonly increased.¹⁸ The LMWOAs enter the soil, which obviously alters the bioavailability of heavy metals in soils and influences their absorption and accumulation by plants.^19–21 Therefore, LMWOAs play an important role in the heavy metal accumulation process in plants.^22–24 For example, citric acid increased the contents of available heavy metals in soil samples and enhanced the accumulation of heavy metals in plants.^25–27 Moreover, the presence of citric acid, malic acid and acetic acid alleviated the biotoxicity of Cd on plants, resulting in an increase in the Cd accumulation of plants.^23,28 Some studies found that oxalic acid caused a mobilization of Cu and Zn in soil, whereas others found that citric acid led to the immobilization of Pb in soil and reduced the absorption and accumulation of heavy metals in plants.^22,29 However, the role of LMWOAs secreted by plant roots in the intercropping of accumulators and crops remains unclear.

Yunnan Province is an important Pb–Zn mine production base in southwest China. Long-term exploitation and smelting of the Pb–zinc mine has resulted in serious Cd and Pb pollution in the farmlands surrounding the mining area.^30–32 In addition, there are large numbers of mining wastelands that feature high heavy metal contents and poor soil fertility and only a minority of plant species are able to grow in these wastelands. Among these plants, some are classified as accumulators and have the capability of enriching a large amount of heavy metals, which provides local plant resources for the remediation of heavy metal-polluted soils.³³ Vicia faba L. is a major winter crop cultivated in the farmlands around the Pb–Zn mine area in Yunnan Province. Due to the great variations in climate across China, agricultural production modes and plant species also vary across the different regions of China. Consequently, each type of accumulator and intercropping programme for soil remediation can only be applied to a specific region and not to the whole country. Furthermore, there are problems associated with the introduction of exotic accumulators to local areas, such as a lack of adaptation to local soil and climatic conditions and the invasion of alien species.¹⁵ Therefore, it is necessary to adopt the local accumulator resources to establish suitable local accumulators and a crops intercropping pattern for remediation on heavy metal-polluted farmlands.

According to natural plant resources and the agricultural production mode in the Huize Pb–Zn mine area, Yunnan Province, we established an intercropping pattern during the summer consisting of a local Cd accumulator, Sonchus asper L., and maize (Zea mays). The S. asper/maize intercropping pattern obviously reduced the amount of Cd and Pb in the maize plants and grains and enhanced Cd and Pb accumulation in S. asper in both pot and field experiments.^34,35 We then established a similar intercropping pattern consisting of S. asper and V. faba during the winter and conducted a field experiment. Taking both S. asper and V. faba monocultures as controls, the effects of S. asper/V. faba intercropping on the plant biomass, LMWOA secretion by plant roots, the contents of available Cd and Pb in soils, and the contents and accumulation of Cd and Pb in plants at 35, 80 and 180 d after planting were investigated. We assumed that (1) intercropping had advantages on promoting the remediation capacity of the accumulator, S. asper, reducing the Cd and Pb contents in crops and enhancing the quality and nutritional safety of the V. faba grains; and (2) the LMWOAs secreted by the intercropping plant roots played an important role in influencing the bioavailability of Cd and Pb in the soil during the intercropping remediation process.

2. Materials and methods

2.1 Experimental field

The experimental field was located at Maseka Village (E 103°38′12.9′′, N 26°34′21.1′′, and altitude 2130 m), Huize County, Yunnan Province in Southwest China. The annual average temperature was 12.6 °C and the annual precipitation was 840 mm. The soil type was mountain red soil. Its physiochemical properties included the following: a pH of 6.11, organic matter content of 21.8 g kg⁻¹, total N, P and K contents of 1.54, 1.75 and 7.44 g kg⁻¹, respectively, available N, P and K contents of 38.1, 71.4 and 614.3 mg kg⁻¹, respectively, and total Cd and Pb contents of 4.59 and 392 mg kg⁻¹, respectively.

2.2 Experimental design

A local Cd accumulator, S. asper, and a main winter crop, V. faba (variety name: Manila), were planted in the experimental farmland. Seeds of S. asper were collected from the Pb–Zn mining area in Huize County, Yunnan Province. The seeds were disinfected with 10% H₂O₂ for 30 min and were then sown into a floating plate filled with a flue-cured tobacco-type matrix. After the S. asper seedlings grew to 5–6 cm, the seedlings and the V. faba seeds were simultaneously transplanted into the farmlands on 5th October. Both the S. asper and V. faba plants were grown from October 2014 to April 2015.

The three planting patterns included a S. asper monoculture, a V. faba monoculture and a S. asper/V. faba intercrop. For the S. asper monoculture, both the between-plant and between-row spaces were 10 cm. For the V. faba monoculture, the between-plant and between-row spacings were 20 and 30 cm, respectively. The intercropping pattern consisted of V. faba rows with intervals of two S. asper rows. For the S. asper/V. faba intercrop, the between-plant space for V. faba was 20 cm; both the between-plant and between-row spacings for S. asper were 10 cm and the row space between S. asper and V. faba was 10 cm. Each planting pattern had three plots and there were a total of nine plots. The size of each plot was 3.0 m × 2 m and they were randomly arranged in the field.

2.3 Sample collection and biomass measurement

Sampling was done at 35 (seedling), 80 (flowering) and 180 d (maturation) after the transplantation of V. faba. Then, three plants planted in the soil of each monoculture plot and three plants of V. faba and S. asper planted in the intercropping plot were randomly chosen and removed from the field. The plants with soil attached to the roots were brought back to the laboratory.

The S. asper plants were divided into underground (roots) and aboveground (stems and leaves) sections. The V. faba plants were divided into three parts (roots, stems and leaves) at both the seedling and flowering stages; the plants were divided into five parts (roots, stems, leaves, pods and grains) when they were in the maturation stage. All the plant parts were washed with tap water and deionized water three times. The plant parts were placed in a drying oven at 105 °C for 30 min to deactivate the enzymes. Then, the samples were dried at 75 °C for 72 h to obtain consistent masses and the biomass of the different plant parts was then measured.

2.4 Measurement of the Cd and Pb contents in the soil and plant samples

The dried plant samples were ground with a pulverizer and separated by a sieve with a pore size of 0.25 mm. The roots were shaken to obtain the attached soil and then the soil was kept out of the sun and dried naturally by indoor air. After fully mixing the soil samples, one part of the soil sample was sized using a sieve with a pore size of 0.25 mm to measure the total contents of Cd and Pb; the remaining soil was sieved with a pore size of 2 mm to measure the available contents of Cd and Pb.

Both the soil and plant samples were digested through wet digestion according to the method published by Bao (2000).³⁶ For the soil samples, 5.0 g portions of the air-dried soil samples that had been subjected to a 0.25 mm nylon sieve were placed in a 150 mL conical flask. A small quantity of water was used to moisten the soil samples. Then, 10 mL of aqua regia (V (concentrated nitric acid) : V (concentrated hydrochloric acid) = 1 : 3) was added into the conical flask and the sample was heated at a low temperature to a slight boiling state (140–160 °C) using an electric heating plate. After the brown nitric oxides almost dried up, the sample was removed from the heating plate and cooled. The addition of perchloric acid (5–10 mL) along the flask wall continued to heat and digest the samples until they turned into a grey white paste. The flask was removed for cooling and distilled water was used to filter the samples into a volumetric flask and to obtain a final volume of 50 mL. For the plant samples, 0.5 g portions of the plants were weighed, digested using the same method as the wet digestion of the soil samples, and the volume was fixed to 50 mL using the distilled water.

The available Cd and Pb contents in the soil samples were determined using a method reported by Bao (2000).³⁶ Air-dried soil samples (25.0 g) that had been filtered through a 0.25 mm sieve were transferred into a conical flask (150 mL) and 20 mL of diethylene triamine penta-acetic acid–tris(2-hydroxyethyl)amine (DTPA–TEA) was added as an extracting agent. The flasks were shaken at 180 rpm for 2 h to extract the available Cd and Pb from the samples. Then, the extract was filtered into a volumetric flask and fixed to a volume of 50 mL with the distilled water.

The Pb concentrations of the solutions in the 50 mL volumetric flasks were determined using flame atomic absorption spectrometry and the Cd concentrations of the samples were measured using graphite furnace atomic absorption spectrometry. Finally, all the Cd and Pb contents in the plants and the total and available contents of Cd and Pb in the soils were calculated using a formula.

2.5 Measurement of LMWOA content secreted by plant roots

After washing off the attached soil with tap water, the plant roots of both V. faba and S. asper were rinsed four times with distilled water. The clean roots were soaked in a 5 mg L⁻¹ methyl propyl phenol solution for 5 min and were then transferred to a collection vessel filled with 300 mL of a CaCl₂ solution (0.5 mmol L⁻¹). The plant roots were placed in the vessel filled with the CaCl₂ solution and the vessel was covered with black plastic to prevent the roots from being exposed to light. The stems and leaves were on the top of the vessel and were kept undisturbed under natural light conditions for 2 h. After removal of the plant roots, the solution was filtered through a 0.45 μm filter to remove the root debris and collect the root exudates. The collected exudates were concentrated to a volume of 3 mL by rotary evaporation at 40 °C. Finally, the concentrations of LMWOAs (oxalic acid, tartaric acid, citric acid, malic acid and lactic acid) in the exudates were determined according to the method published by Cawthray (2003) with minor modifications.³⁷ The amounts of LMWOAs were measured by high performance liquid chromatography (HPLC) with an Agilent 20RBAX SB-C18 (250 × 4.6 mm ID) column. The mobile phase was 2% methanol at a flow rate of 0.6 mL min⁻¹; a 15 μL sample volume was loaded, the peaks were detected at 210 nm, and the analysis time was 40 min.

2.6 Measurement of the accumulation features of Cd and Pb

The accumulation amounts of Cd and Pb in the plants was the sum of the Cd and Pb contents multiplied by the plants' biomass. The accumulation features of Cd and Pb were expressed through an enrichment coefficient (EC) and a translation coefficient (TC). EC = the content of Cd and Pb in the aboveground parts/their contents in the soil. TC = the content of Cd and Pb in the aboveground parts/their contents in the underground parts.²⁸

2.7 Data and statistical analyses

Preliminary data processing was performed in Excel 2010. Significant differences between the treatments were determined using the independent samples T-test. The correlations between the contents of the LMWOAs secreted by the plant roots, the contents of available Cd and Pb in the soil, between the contents of the available Cd and Pb in the soil and the Cd and Pb contents in each part of the plants were determined using the respective functions in SPSS 22.0.

3. Results and discussion

3.1 Plant biomass

The plant biomass of V. faba and S. asper increased with the progress of plant growth. For V. faba, no significant difference was found on the biomass of the different plant parts (roots, stems, leaves, pods and grains) between those grown in monocultures and intercrops; however, the stem biomass of the plants grown in a monoculture was significantly higher than those grown in intercrops at 80 d after planting (Fig. 1).

Fig. 1 Biomass of V. faba in the monoculture and intercropping. All values represent the mean ± standard error (SE), n = 9. “**” means very significant difference (P < 0.01).

For S. asper, there were no significant differences between plants grown in a monoculture and intercrop on the roots biomass at all the three stages or on the stem and leaf biomass at 30 and 80 d after planting. However, the stem and leaf biomass of plants grown in an intercrop were significantly higher than those grown in a monoculture at 180 d after planting (Fig. 2).

Fig. 2 Biomass of S. asper in the monoculture and intercropping. All values represent the mean ± standard error (SE), n = 9. “*” means significant difference (P < 0.05).

However, the effects of intercropping different accumulators (or hyperaccumulators) and crops on the contents and accumulation of heavy metals by crops were different. Some studies have found that intercropping reduced the contents of heavy metals in plants and grains and increased the amount of biomass and production of crops. For example, the intercropping of plants, such as Thlaspi arvense (a Zn hyperaccumulator)/barley,³⁸ T. arvense/Chinese cabbage,³⁹ Sedum alfredii (a Zn hyperaccumulator)/maize,^10,15 Conyza canadensis (a Cd hyperaccumulator)/cherry seedling, Solanum nigrum (a Cd hyperaccumulator)/cherry seedling, Digitaria sanguinalis (a Cd and Pb hyperaccumulator)/cherry seedling,⁴⁰ Pteris vittata (a As hyperaccumulator)/Panax notoginseng,¹³ Brassica juncea (a Cd hyperaccumulator)/alfalfa,¹² and Thalia dealbata/rice,⁴¹ reduced heavy metal (Zn, Pb, Cd, Cu and As) contents in the plants and gains of crops and increased the biomass of crops. Similar to these study results, this study found that S. asper/V. faba intercropping resulted in a decrease in the Cd and Pb contents in V. faba and an increase in both the contents and accumulation of Cd and Pb in S. asper.

Some other studies have reported that the intercropping of accumulators (or hyperaccumulators) and crops did not have a notable influence and even demonstrated that it increased the contents of heavy metals in the plants and grains of crops. For instance, S. nigrum/onion intercropping was shown to have no influence on the Cd content in onion plants under field conditions.⁹ B. juncea/oilseed rape intercropping caused an increase in the Cd content in the plant and a decrease in the yield of oilseed rape.⁴² Pteris cretica (an As hyperaccumulator)/maize intercropping even resulted in an increase in the contents of As, Pb and Cd in the roots, stems and leaves of maize.⁴³ Sedum plumbizincicola (a Cd hyperaccumulator)/wheat intercropping also increased the Zn and Cd contents in the aboveground parts of wheat.⁴⁴

3.2 LMWOAs secreted by plant roots

The main LMWOAs secreted by V. faba roots systems were oxalic acid, tartaric acid and citric acid at 35 d after planting; citric acid was detected with the progress of plant growth. However, the main LMWOAs secreted by the S. asper roots were oxalic acid, tartaric acid and citric acid at the three growth stages.

Intercropping altered the secretion of LMWOAs by the V. faba and S. asper roots. For V. faba, intercropping led to a significant increase in the contents of citric acid at 35 d, lactic acid at 80 d, and tartaric acid at 180 d; moreover, a significant decrease in lactic acid was observed at 35 d after planting. For S. asper, intercropping caused a significant decrease in the contents of citric acid and lactic acid at 35 d, oxalic acid, tartaric acid and citric acid at 80 d, and citric acid at 180 d after planting. Overall, compared to the plants grown in monocultures, intercropping resulted in an increase and decrease in the LMWOAs contents secreted by V. faba and S. asper roots, respectively (Fig. 3).

Fig. 3 Exudation of LMWOAs secreted by V. faba and S. asper roots in the monoculture and intercropping. Oxalic acid (OA), tartaric acid (TA), citric acid (CA), malic acid (MA) and lactic acid (LA). All values represent the mean ± standard error (SE), n = 3. “*” means significant difference (P < 0.05).

In fact, the intercropping influenced the roots' LMWOAs exudation, as reported by some studies,^45,46 such as with the intercropping of faba bean with maize, which resulted in the amount of malic acid exuded by intercropped faba bean being higher than with monocropped plants.⁴⁵ However, the mechanisms for the changes of the LMWOAs exudation induced by the intercropping are still unclear.

3.3 Contents of available Cd and Pb in soil

In the present study, the data refer to the rhizosphere soil adherent to the roots surface and it's a layer about 1–2 mm thick. The rhizosphere soil is significantly influenced by the root exudates secreted into the rhizosphere. Moreover, the effects of root exudates on the soils chemistry decreased in the bulk soil.

Both at 35 and 80 d after planting, the available Cd and Pb contents were less in the soils of the S. asper monoculture and were higher in the soils of the V. faba monoculture and the S. asper/V. faba intercrop. However, at 180 d after planting, the available Cd and Pb contents were smallest in the soils of the S. asper/V. faba intercrop and were very significantly less than samples from both the V. faba and S. asper monocultures (Fig. 4).

Fig. 4 Available Cd content in the soils in the V. faba monoculture, S. asper monoculture and S. asper/V. faba intercropping. All values represent the mean ± standard error (SE), n = 9. Different capital letters mean very significant difference (P < 0.01), different lowercase letters mean significant difference (P < 0.05).

With the plants growth progress, both the available Cd and Pb contents decreased in the soils for the S. asper/V. faba intercrop but increased for the S. asper monoculture. For the V. faba monoculture, there was a decrease in the available Cd content and an unnoticeable change in the available Pb content in the soils (Fig. 4). Therefore, the S. asper/V. faba intercrop resulted in a decrease in the availability of both the Cd and Pb in the soils.

3.4 Accumulation features of Cd and Pb in plants

Compared with the V. faba monoculture, the intercropping led to a significant decrease in the Cd content in the roots and grains at 180 d, and a significant decrease in the stems at 80 d after planting. In addition, the intercropping resulted in significant decreases in the Pb content in the roots at 80 d and the stems at 35 and 180 d after planting (Table 1). Hence, intercropping resulted in a decrease in the Cd and Pb contents in the plants and grains of V. faba.

Table 1 Contents (mg kg⁻¹) of Cd and Pb in monoculture and intercropping plants of V. faba^a

Heavy metals	Plant parts	Planting pattern	35 d after planting	80 d after planting	180 d after planting
a All values represent the mean ± standard error (SE), n = 9. “**” means very significant difference (P < 0.01), and “*” means significant difference (P < 0.05) between monoculture and intercropping. “—” means without the plant parts at the sampling time.
Cd	Roots	Monoculture	5.67 ± 0.41	5.37 ± 0.31	5.19 ± 0.32*
		Intercropping	4.91 ± 0.27	5.19 ± 0.39	4.30 ± 0.25
	Stems	Monoculture	3.25 ± 0.32	4.62 ± 0.36	3.69 ± 0.43
		Intercropping	2.73 ± 0.29	4.77 ± 0.32	3.23 ± 0.28
	Leaves	Monoculture	2.72 ± 0.09	4.56 ± 0.22**	3.95 ± 0.42
		Intercropping	3.47 ± 0.42	2.92 ± 0.36	3.42 ± 0.32
	Pods	Monoculture	—	—	3.60 ± 0.24
		Intercropping	—	—	2.96 ± 0.20
	Grains	Monoculture	—	—	1.08 ± 0.09*
		Intercropping	—	—	0.72 ± 0.09
Pb	Roots	Monoculture	154.5 ± 9.3	131.0 ± 8.3*	136.8 ± 9.3
		Intercropping	143.6 ± 4.2	107.7 ± 6.8	136.9 ± 11.8
	Stems	Monoculture	73.9 ± 5.6*	92.2 ± 6.2	146.9 ± 8.9*
		Intercropping	55.8 ± 2.9	98.5 ± 8.3	116.2 ± 6.0
	Leaves	Monoculture	52.6 ± 4.4	101.1 ± 7.5	110.2 ± 3.9
		Intercropping	56.2 ± 2.1	109.4 ± 4.6	100.4 ± 2.7
	Pods	Monoculture	—	—	98.0 ± 5.5
		Intercropping	—	—	90.2 ± 5.4
	Grains	Monoculture	—	—	50.2 ± 4.3
		Intercropping	—	—	44.4 ± 3.1

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Furthermore, the intercropping led to a significant decrease in the Cd accumulation in the leaves at 80 d and 180 d after planting and a very significant decrease in the pods at 180 d after planting; it also resulted in significant decreases in the Pb accumulation in the roots (at 80 d), stems (at 35 and 80 d) and pods (at 180 d), and a very significant decrease in the stems at 180 d after planting (Table 2). Therefore, the intercropping also resulted in a decrease in Cd and Pb accumulation in the plants and grains of V. faba.

Table 2 Accumulation (ng per plant) of Cd and Pb in monoculture and intercropping plants of V. faba^a

Heavy metals	Plant parts	Planting pattern	35 d after planting	80 d after planting	180 d after planting
a All values represent the mean ± standard error (SE), n = 9. “**” means very significant difference (P < 0.01), “*” means significant difference (P < 0.05) between monoculture and intercropping. “—” means without the plant parts at the sampling time.
Cd	Roots	Monoculture	2.6 ± 0.2	8.0 ± 0.5	14.9 ± 1.2
		Intercropping	2.2 ± 0.2	6.7 ± 0.8	11.9 ± 1.4
	Stems	Monoculture	1.3 ± 0.2	8.9 ± 1.3	40.7 ± 3.7
		Intercropping	0.9 ± 0.1	6.3 ± 0.4	32.5 ± 3.7
	Leaves	Monoculture	3.6 ± 0.5	14.7 ± 1.1*	19.4 ± 2.7*
		Intercropping	4.2 ± 0.6	9.9 ± 1.5	17.7 ± 2.0
	Pods	Monoculture	—	—	20.9 ± 1.5**
		Intercropping	—	—	15.1 ± 1.8
	Grains	Monoculture	—	—	10.2 ± 0.5
		Intercropping	—	—	5.9 ± 0.8
Pb	Roots	Monoculture	72 ± 8	195 ± 14*	393 ± 33
		Intercropping	66 ± 6	138 ± 14	384 ± 54
	Stems	Monoculture	27 ± 4*	168 ± 9*	1675 ± 138**
		Intercropping	18 ± 1	131 ± 12	1158 ± 86
	Leaves	Monoculture	71 ± 14	326 ± 30	534 ± 43
		Intercropping	67 ± 5	364 ± 32	515 ± 28
	Pods	Monoculture	—	—	570 ± 37*
		Intercropping	—	—	440 ± 27
	Grains	Monoculture	—	—	496 ± 64
		Intercropping	—	—	363 ± 32

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In contrast, intercropping resulted in an increase in both the contents and accumulation of Cd and Pb in the stems and leaves of S. asper. Compared with a S. asper monoculture, the Cd contents in the stems and leaves obviously increased in the three stages, and Cd accumulation in the stems and leaves increased significantly or very significantly at 35, 80 and 180 d after planting. A very significant increase in the Pb content at 35 d and a significant increase in the accumulation of Pb in roots, stems and leaves at 180 d after planting were observed (Tables 3 and 4).

Table 3 Contents (mg kg⁻¹) of Cd and Pb in monoculture and intercropping plants of S. asper^a

Heavy metals	Plant parts	Planting pattern	35 d after planting	80 d after planting	180 d after planting
a All values represent the mean ± standard error (SE), n = 9. “**” means very significant difference (P < 0.01) between monoculture and intercropping.
Cd	Roots	Monoculture	4.96 ± 0.35	4.80 ± 0.34	9.33 ± 1.58
		Intercropping	4.00 ± 0.27	5.83 ± 0.53	9.85 ± 1.08
	Stems and leaves	Monoculture	3.40 ± 0.26	4.15 ± 0.62	6.63 ± 0.52
		Intercropping	5.40 ± 0.35**	6.57 ± 0.37**	10.30 ± 0.86**
Pb	Roots	Monoculture	104.1 ± 6.9	233.8 ± 15.9	364.1 ± 32.9
		Intercropping	97.8 ± 5.7	208.8 ± 11.6	449.2 ± 36.9
	Stems and leaves	Monoculture	41.9 ± 1.7	184.4 ± 16.3	386.3 ± 31.4
		Intercropping	58.0 ± 1.3**	215.0 ± 19.0	424.5 ± 30.2

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Table 4 Accumulation (ng per plant) of Cd and Pb in monoculture and intercropping plants of S. asper^a

Heavy metals	Plant parts	Planting pattern	35 d after planting	80 d after planting	180 d after planting
a All values represent the mean ± standard error (SE), n = 9. “**” means very significant difference (P < 0.01), and “*” means significant difference (P < 0.05) between monoculture and intercropping.
Cd	Roots	Monoculture	2.3 ± 0.3	7.6 ± 0.7	11.7 ± 1.3
		Intercropping	1.8 ± 0.3	8.6 ± 1.0	15.6 ± 2.2
	Stems and leaves	Monoculture	2.2 ± 0.4	6.6 ± 0.8	26.5 ± 2.2
		Intercropping	3.4 ± 0.4*	11.2 ± 0.7**	48.9 ± 3.1**
Pb	Roots	Monoculture	48 ± 5	366 ± 24	489 ± 48
		Intercropping	43 ± 6	308 ± 25	696 ± 72*
	Stems and leaves	Monoculture	27 ± 3	299 ± 20	1537 ± 133
		Intercropping	38 ± 5	359 ± 28	2083 ± 207*

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As shown in Table 5, S. asper/V. faba intercropping obviously increased the enrichment coefficient (EC) and translation coefficient (TC) of Cd for S. asper at the three stages compared with the S. asper monoculture, with the highest remediation efficiency detected at 180 d after planting. In contrast, there was a very small increase in both the EC and TC of Pb. For V. faba, there were no big differences in both the EC and TC of the Cd and Pb between the monoculture and intercropping. These results indicated that intercropping promoted Cd translation from the roots to the stems and leaves and enhanced the Cd accumulation of S. asper.

Table 5 Enrichment coefficient (EC) and translation coefficient (TC) of Cd and Pb for S. asper and V. faba

Plant	Planting time (d)	Planting pattern	Cd		Pb
			EC	TC	EC	TC
S. asper	35	Monoculture	0.74	0.69	0.11	0.40
		Intercropping	1.18	1.35	0.15	0.59
	80	Monoculture	0.90	0.86	0.47	0.79
		Intercropping	1.43	1.13	0.55	1.03
	180	Monoculture	1.44	0.71	0.99	1.06
		Intercropping	2.24	1.05	1.08	0.95
V. faba	35	Monoculture	0.62	0.50	0.15	0.37
		Intercropping	0.72	0.67	0.14	0.39
	80	Monoculture	1.00	0.85	0.25	0.75
		Intercropping	0.75	0.66	0.27	0.99
	180	Monoculture	0.63	0.56	0.26	0.75
		Intercropping	0.54	0.58	0.22	0.64

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3.5 Correlation analyses

Correlation analyses were conducted between the LMWOAs secreted by the plant roots and the available Cd and Pb contents in the soils. Significant negative correlations were observed between the contents of oxalic acid and tartaric acid secreted by the S. asper roots, citric acid and malic acid secreted by the V. faba roots and the contents of available Cd in the soils; their correlation coefficients were −0.541 (n = 18), −0.462 (n = 18), −0.534 (n = 18), and −0.578 (n = 15). However, there was no significant correlation between the LMWOAs contents and the available Pb contents (Table 6). These results indicated that the LMWOAs secreted by the roots of S. asper and V. faba had obvious effects on reducing the availability of Cd in the soils.

Table 6 Correlation coefficient between the LMWOAs and both the available Cd and Pb contents in the soils^a

Heavy metals	Plant	Oxalic acid	Tartaric acid	Citric acid	Malic acid	Lactic acid
a “*” means significant difference (P < 0.05).
Available Cd	S. asper	−0.541*	−0.462*	−0.366	−0.058	0.164
	V. faba	0.240	0.193	−0.534*	−0.578*	−0.150
Available Pb	S. asper	−0.042	0.013	−0.163	−0.118	0.102
	V. faba	0.182	0.169	−0.083	−0.351	−0.209

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The LMWOAs excreted by intercropping plants had notable effects on the heavy metals bioavailability in soils and on the uptake by the plants. For example, in a barley/pea intercropping system, intercropping promoted peas to accumulate heavy metals and this observation was related to the mobilization of heavy metals in soil by the root exudates of the intercropped barley.⁴⁷ The present study found that the LMWOAs secreted by the roots of S. asper and V. faba in the intercropping system reduced the contents of available Cd in soils, and the intercropping reduced the Cd content in the plants and grains of V. faba. Furthermore, a significant positive correlation was observed between the content of available Cd in soils and the Cd contents in the roots and grains of V. faba. Therefore, the functional mechanism of intercropping and its influence on the accumulation of heavy metals in plants was related to the LMWOAs secreted by plant roots and their effects on the bioavailability of heavy metals in soil samples.

Furthermore, a correlation analysis was conducted between the available Cd and Pb contents in soils and the Cd and Pb contents in different parts of the plants. Significant positive correlations were observed between the content of available Cd in the soil samples and the Cd contents in the roots and grains of V. faba; their correlation coefficients were 0.488 (n = 18) and 0.835 (n = 6), respectively.

Summing up the correlations of the secretion of LMWOAs, the available Cd and Pb contents in the soil samples and the Cd and Pb contents in plants, our observations indicated that intercropping reduced the Cd contents in the roots and grains of V. faba, which was closely related to the effects of reducing the amount of LMWOAs secreted by plant roots on the availability of Cd in soil.

In addition, the interspecific root interactions between plants in an intercropping system were also shown to play a significant role in the interactive effects of intercropping plants. This included the root-system spatial distribution heterogeneity caused by the recognition behaviour of roots and the morphology between the roots (i.e., “root–root”). In addition, the biological behavior of roots in “root–root symbiont–root” systems was found to be mediated by the root symbiont.^48,49 In heavy metal-polluted soils, intercropping led to a change in the soils' physicochemical properties, contents of available heavy metals and on the translation of heavy metals from soils to plants. Its underground mechanism for reducing the accumulation of heavy metals by crops included the foraging and high absorption of heavy metals in soils by hyperaccumulators,⁵⁰ and the symbiotic effects of arbuscular mycorrhizal fungi in plant roots.¹¹ Finally, the influencing mechanisms of intercropping on the absorption and accumulation of heavy metals by hyperaccumulators and crops are still unclear and need to be further studied.

Finally, S. asper/V. faba intercropping presented an outstanding effect on decreasing the Cd and Pb contents in the plants and grains of V. faba and on enhancing the accumulation of Cd and Pb in S. asper under field conditions. It can be noted that the remediation efficiency of S. asper was the highest at 180 d after planting, which was also the harvest time for V. faba. Therefore, both S. asper and V. faba were harvested simultaneously with significant regional advantages, which included: (1) the local accumulator had adapted to the local soil and climate conditions, thus avoiding problems that might be faced by an exotic accumulator such as environmental inadaptation and the invasion threats of alien species. (2) The wild S. asper seeds in the Pb–Zn mining area were abundant and used to cultivate a large quantity of seedlings to meet the seedling demand for building the intercropping system in the field pattern, thus realizing continuous remediation of the polluted soil under field conditions. (3) Because this intercropping remediation did not alter the local planting modes and habits and because the accumulator and crop were harvested at the same time, local farmers would more easily accept and apply this method to simultaneously achieve both remediation and agricultural production. However, some problems still exist, including the limited remediation efficiency, the contents of heavy metals in crop grains still exceeded the hygienic standard limits for agricultural products, and difficulties of applying intercropping remediation in field with agricultural machinery. Thus, more studies need to be conducted on the remediation mechanisms and applications of intercropping methodologies.

4. Conclusions

Under field conditions, intercropping of the Cd accumulator S. asper/V. faba resulted in a decrease in the Cd and Pb contents in the plants and grains of V. faba and an increase in the biomass and the Cd and Pb contents in S. asper. Intercropping provided a new feasible way for both improving the safety of agricultural products and enhancing the remediation efficiency of accumulators on polluted farmlands. The major LMWOAs secreted by both S. asper and V. faba were oxalic acid, tartaric acid and citric acid. Intercropping resulted in an increase and a decrease in the LMWOAs secreted by V. faba and S. asper roots, respectively; and a decrease in the contents of available Cd and Pb in soils was observed. There were significant negative correlations between the contents of citric acid, malic acid (secreted by V. faba roots), oxalic acid and tartaric acid (secreted by S. asper roots) with the available Cd content in the soil. A significant positive correlation was observed between the available Cd content in the soil and the Cd content in the roots and grains of V. faba. These results indicated that the mechanism of intercropping reduced the Cd content in V. faba was closely related with the bioavailability of Cd in soils mediated by LMWOAs secreted from intercropping plant roots.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (No. U1202236, 41461093, 41261096 and 31560163).

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