Effects of Mulching Tolerant Plant Straw on Soil Surface on Growth and Cadmium Accumulation of Galinsoga parviflora

Pot and field experiments were conducted to study the effects of mulching with straw of cadmium (Cd) tolerant plants (Ranunculus sieboldii, Mazus japonicus, Clinopodium confine and Plantago asiatica) on growth and Cd accumulation of Galinsoga parviflora in Cd-contaminated soil. In the pot experiment, mulching with M. japonicus straw increased the root biomass, stem biomass, leaf biomass, shoot biomass, plant height and activities of antioxidant enzymes (superoxide dismutase, peroxidase and catalase) of G. parviflora compared with the control, whereas mulching with straws of R. sieboldii, C. confine and P. asiatica decreased these parameters. Straws of the four Cd-tolerant plants increased the Cd content in roots of G. parviflora compared with the control. However, only straws of M. japonicus and P. asiatica increased the Cd content in shoots of G. parviflora, reduced the soil pH, and increased the soil exchangeable Cd concentration. Straw of M. japonicus increased the amount of Cd extraction in stems, leaves and shoots of G. parviflora by 21.11%, 29.43% and 24.22%, respectively, compared with the control, whereas straws of the other three Cd-tolerant plants decreased these parameters. In the field experiment, the M. japonicus straw also increased shoot biomass, Cd content in shoots, and amount of Cd extraction in shoots of G. parviflora compared with the control. Therefore, straw of M. japonicus can be used to improve the Cd extraction ability of G. parviflora from Cd-contaminated soil.


Introduction
As anthropogenic impacts on the environment continue to intensify, increasing soil contamination with heavy metals has become a major factor harming human health [1]. In China, the area of cadmium (Cd) contaminated soil has increased to 200,000 km 2 , which is one-sixth of the arable land area [2]. Therefore, the heavymetal-contaminated soils require immediate treatment, especially for Cd. A variety of methods are suitable for remediation of heavy-metal-contaminated soil, of which phytoremediation is a commonly used method [3]. Phytoremediation maintains the soil structure and microbial community, while the roots of hyperaccumulator plants directly absorb heavy metals from the soil and transfer them to the aboveground shoots to achieve remediation of contaminated soil [4]. However, the main hyperaccumulator materials used for phytoremediation are slow-growing and produce low biomass, thus large-scale application is difficult [5][6]. Therefore, improvement in the remediation ability of hyperaccumulator is of practical importance.
Methods that can improve the remediation ability of hyperaccumulators have been screened, such as application of surfactant chelator to the soil [7], intercropping [8][9], and spray application of a plant growth regulator [10][11]. While these measures have been beneficial to a certain degree, their application has not been widely implemented. As diverse agricultural production practices are used in different areas, screening of more natural methods to improve remediation ability is necessary. Straw mulching is a technique that is commonly used in agricultural production. The main purpose of the straw mulch is the conversion of plant straw into organic matter and other nutrients, and to fertilize the soil and improve soil texture [12][13]. The straw can improve the physical and chemical properties of soil, increase soil organic matter content, and therefore promote crop growth, increase yield, and improve crop quality [14]. These effects are related to the nutrients and allelochemicals released from the straw. Release of allelochemicals is a form of allelopathy, which can affect soil nutrient availability, soil enzyme activity, microbial population structure, and plant growth [15]. If plant straw was applied to heavy metal-contaminated soil, allelochemicals may regulate hyperaccumulator growth and heavy metal accumulation. As Cd-tolerant plants, Ranunculus sieboldii, Clinopodium confine, Mazus japonicus and Plantago asiatica show high tolerance to Cd stress, and Cd contents in the shoots of these species are much lower than those of a hyperaccumulator or accumulator [16]. In this study, we grew seedlings of the Cd hyperaccumulator Galinsoga parviflora [16] in Cd-contaminated soil. Shoots of the above-mentioned Cd-tolerant species were applied as mulches on the soil surface to screen their efficiency at promoting the growth and Cd accumulation of G. parviflora, and to provide materials for improvement in the phytoremediation ability of G. parviflora.

Ethics statement
The field study was conducted in an area where Ranunculus sieboldii, Clinopodium confine, Mazus japonicus, Plantago asiatica and Galinsoga parviflora were the main weeds. This site was on the Ya'an campus of Sichuan Agricultural University Farm (29˚599N, 102˚599E), Ya'an City, Sichuan Province, China. The experimental field belongs to Sichuan Agricultural University, which is authorized by the government of Ya'an City. The field is not privately owned or protected. No specific permits were required for the described field studies. During the experiment, no other specific permissions were required because we conducted normal agricultural activities and no endangered or protected species were involved.

Plant and soil materials
In August 2013, shoots of Ranunculus sieboldii, Clinopodium confine, Mazus japonicus and Plantago asiatica were collected from the Ya'an campus farm of the Sichuan Agricultural University (29˚599 N, 102˚599 E), China, at sites where the soil was not contaminated by heavy metals. The shoots were dried at 80˚C to constant weight, then finely ground and sieved through a 5-mm-mesh nylon sieve. Galinsoga parviflora seedlings with two pairs of euphyllas were collected from the Ya'an campus farm at a site not contaminated by heavy metals in September 2013.
Inceptisol soil samples (purple soil in the Genetic Soil Classification of China) were collected from the Ya'an campus farm in August 2013. The basic chemical properties of the soil were pH 7.02, organic matter 41.38 g kg 21 , total nitrogen (N) 3.05 g kg 21 , total phosphorus (P) 0.31 g kg 21 , total potassium (K) 15.22 g kg 21 , alkali soluble N 165.30 mg kg 21 , available P 5.87 mg kg 21 , and available K 187.03 mg kg 21 . The total Cd content was 0.101 mg kg 21 and the available Cd content was 0.021 mg kg 21 . The basic soil properties and heavy metal concentrations were determined according to Bao (2000) [17].

Pot experiment
The experiment was conducted at the Ya'an campus farm in August-October 2013. The soil samples were air-dried and passed through a 5-mm sieve in August 2013, and then 4.0 kg of the air-dried soil was weighed into each polyethylene pot (15 cm high, 18 cm diameter). Cadmium was added to the soil samples as CdCl 2 ?2.5 H 2 O at 10 mg kg 21 [18]. The pots were soaked in the Cd solution for 4 weeks, and then the soil in each pot was mixed thoroughly. Five uniform seedlings of G. parviflora were transplanted into each pot, and 6 g shoots [19] of four Cdtolerant species were applied as mulches on the soil surface in each pot (equivalent to 225 g m 22 ). Five treatments were applied: not mulched with straw (control), mulched with R. sieboldii straw, mulched with C. confine straw, mulched with M. japonicus straw, and mulched with P. asiatica straw. Each treatment was repeated three times with a completely randomized design with 10-cm spacing between pots. The soil moisture was maintained at 80% of field capacity from when the G. parviflora plants were transplanted until the plants were harvested.
At maturity (after 50 d), the height of the G. parviflora plants was measured, and the uppermost young leaves of 2 cm in length were collected to determine the activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) [20]. Then, the entire plants were harvested. The roots, stems, and leaves were washed with tap water, and the roots were immersed in 10 mM L 21 HCl for 10 min to remove Cd adhering to the root surface. The roots, stems, and leaves were further washed with deionized water and dried at 80˚C to constant weight for dry weight and Cd content determination. The dried plant samples were finely ground and sieved through a 0.149-mm-mesh nylon sieve for chemical analysis. Samples (0.5 g) were digested in HNO 3 /HClO 4 (4:1, v/ v), and then the volume was diluted to 50 ml with deionized water. The Cd concentrations in roots, stems, and leaves were determined using an iCAP 6300 ICP spectrometer (Thermo Scientific, Waltham, MA, USA) [17]. The pot soils were dried naturally and ground into powder (granule diameter ,1 mm) to determine the pH and exchangeable Cd concentration. Soil pH was measured in a 1:2.5 (w/v) suspension of soil and deionized water, and the exchangeable Cd in the soil was extracted with 0.005 mol L 21 DTPA-TEA and analyzed with an iCAP 6300 ICP spectrometer [17].

Field experiment
The field experiment was conducted at the Ya'an campus farm in February-March 2014. Inceptisol soil samples were collected from the Cd-contaminated area of the farm. The basic chemical properties of the soil were as follows: pH 6.96, organic matter 34.33 g kg 21 , total N 1.14 g kg 21 , total P 0.66 g kg 21 , total K 21.54 g kg 21 , alkali solution N 78.55 mg kg 21 , available P 33.67 mg kg 21 , available K 111.07 mg kg 21 , and total Cd 1.80 mg kg 21 . The basic soil properties and heavy metal concentrations were determined according to Bao (2000) [17]. Each plot area was 1.0 m 2 (1.0 m61.0 m). Seedlings of G. parviflora were planted directly in the soil at a density of 100 plants m 22 (in a 10 cm610 cm grid) in February 2014. Shoots of the four Cd-tolerant species were applied as mulches (density 225 g m 22 ) on the soil surface in each plot. The five treatments in the experiment were identical to those of the pot experiment. Each treatment was repeated three times (three plots). The cultivation and management of G. parviflora seedlings were as previously described for the pot experiment. At maturity (after 50 d), the G. parviflora shoots were harvested to determine shoot biomass and Cd content as described for the pot experiment.

Statistical analyses
Statistical analyses were performed using SPSS 13.0 statistical software (IBM, Chicago, IL, USA). Data were analyzed by one-way ANOVA with least significant difference (LSD) at the 5% confidence level.

Biomass of G. parviflora
In the pot experiment, the M. japonicus straw mulch significantly (P,0.05) increased the root, stem, leaf, and shoot biomasses of G. parviflora by 18.75%, 9.35%, 33.33%, and 16.11%, respectively, compared with those of the control, whereas mulching with R. sieboldii, C. confine, and P. asiatica straws decreased these biomasses compared with the control ( Table 1). The treatment effects on root biomass of G. parviflora were ranked as M. japonicus straw . control .P. asiatica straw . C. confine straw . R. sieboldii straw, and on stem, leaf and shoot biomass as M. japonicus straw . control . C. confine straw .P. asiatica straw . R. sieboldii straw. Mulching with straws of the four Cd-tolerant species increased the root/shoot ratio of G. parviflora compared with the control, for which the treatment effects were ranked as P. asiatica straw . R. sieboldii straw . C. confine straw . M. japonicus straw . control (Table 1), which indicated that straws of the four Cd-tolerant species promoted G. parviflora root growth.

Antioxidant enzyme activity of G. parviflora
Mazus japonicus straw enhanced the activities of SOD, POD and CAT of G. parviflora in the pot experiment by 8.31% (P,0.05), 9.42% (P,0.05), and 6.68% (P,0.05), respectively, compared with the control, whereas R. sieboldii, C. confine, and P. asiatica straws decreased activities of these enzymes ( Table 2). The treatment effects on SOD, POD and CAT activities of G. parviflora were ranked as M. japonicus straw . control . C. confine straw .P. asiatica straw . R. sieboldii straw, which was consistent with the effects on G. parviflora biomass.

Cadmium content in G. parviflora
Mulching with straws of the four Cd-tolerant species changed the Cd distribution in organs of G. parviflora in the pot experiment. All of the straws increased the Cd content in roots of G. parviflora in the rank order P. asiatica straw . M. japonicus straw . R. sieboldii straw . C. confine straw . control (Table 3). Only the straws of M. japonicus and P. asiatica increased the Cd content in stems of G. parviflora, which increased by 10.75% (P,0.05) and 4.64% (P.0.05), respectively, compared with that of the control, whereas the straws of R. sieboldii and C. confine decreased the stem Cd content. The treatments affected Cd content in leaves of G. parviflora in the rank order P. asiatica straw . R. sieboldii straw . C. confine straw . M. japonicus straw . control. The treatments had a similar effect on Cd content in shoots of G. parviflora to that on stem Cd content. The straws of M. japonicus and P. asiatica increased shoot Cd content by 8.34% (P,0.05) and 13.67% (P,0.05), respectively, compared with the control. The shoot BCF of G. parviflora was enhanced by the straws of M. japonicus and P. asiatica, but was decreased by the straws of R. sieboldii and C. confine, compared with the control ( Table 3). The straw of M. japonica enhanced the TF of G. parviflora, whereas the other straw types decreased the TF, compared with that of the control.

Cadmium extraction by G. parviflora
In the pot experiment, the straws of M. japonicus and P. asiatica increased the amount of Cd extraction in roots of G. parviflora by 27.88% (P,0.05) and 16.68% (P,0.05), respectively, compared with that of the control, whereas the R. sieboldii and C. confine straws decreased root Cd extraction (Table 4). Only M. japonicus straw increased the amount of Cd extraction in stems, leaves, and shoots of G. parviflora by 21.11% (P,0.05), 29.43% (P,0.05), and 24.22% (P,0.05), respectively, compared with the control, whereas the other three straw types decreased Cd extraction in these organs ( Table 4). The treatment effects on the TAF of G. parviflora were ranked as control . M. japonicus straw . C. confine straw . R. sieboldii straw .P. asiatica straw. Soil pH and soil exchangeable Cd The straws of R. sieboldii and C. confine increased the soil pH (P.0.05) compared with that of the control, whereas straws of M. japonicus (P.0.05) and P. asiatica (P,0.05) decreased soil pH in the pot experiment (Fig. 2). The treatment effects on soil pH were ranked as C. confine straw . R. sieboldii straw . control . M. japonicus straw .P. asiatica straw. The effects on soil exchangeable Cd concentration were ranked as P. asiatica straw . M. japonicus straw . control . R. sieboldii straw . C. confine straw in the pot experiment (Fig. 3). The straws of R. sieboldii and C. confine decreased the soil exchangeable Cd concentration by 0.27% (P.0.05) and 1.37% (P,0.05), respectively, whereas the straws of M. japonicus and P. asiatica increased soil exchangeable Cd concentration by 1.09% (P,0.05) and 1.50% (P,0.05), respectively, compared with that of the control.

Field experiment
In the field experiment, M. japonicus straw increased the shoot biomass of G. parviflora compared with that of the control (P,0.05), whereas the other three treatments decreased the shoot biomass (Table 5), which was consistent with the results of the pot experiment. Mulching with the straws of M. japonicus and P. asiatica increased, whereas the straws of R. sieboldii and C. confine decreased the Cd content in shoots of G. parviflora compared with that of the control. Compared with the control, the Cd extraction amount in shoots of G. parviflora was increased by M. japonicus straw by 6.41% (P,0.05), but was decreased by the other three treatments (Table 5).

Discussion
Plant straw contains many nutrients and allelochemicals, which are released during the process of straw decay and decomposition and can be absorbed by living plants [25]. However, when plant straw decays and decomposes, organic acids are produced and can inhibit the growth of living plant roots to some extent, eventually leading to inhibition of plant shoot growth [26]. In the present pot experiment, mulching with M. japonicus straw increased the root, stem, leaf, and shoot biomasses of G. parviflora compared with those of the control, whereas the straws of R. sieboldii, C. confine, and P. asiatica decreased these biomasses. An identical effect on shoot biomass of G. parviflora was observed in the field experiment. There might be two reasons for this phenomenon. First, the  promotive effect of nutrients from M. japonica straw on growth of G. parviflora exceeded the inhibitory effect of organic acids from M. japonica straw, whereas the promotive effect of nutrients from R. sieboldii, C. confine, and P. asiatica straws on growth of G. parviflora was weaker than the inhibitory effect of organic acids from these straws. Second, allelochemicals from M. japonica straw might promote growth of G. parviflora, whereas allelochemicals from R. sieboldii, C. confine, and P. asiatica straws might inhibit growth of G. parviflora. Ultimately, in the pot experiment, M. japonicus straw enhanced, whereas R. sieboldii, C. confine, and P. asiatica straws decreased the plant height of G. parviflora compared with that of the control. Under stress conditions, plants produce O 2 2 ? in their organs, leading to cellular peroxide poisoning and damage, which inhibits plant growth and reduces plant biomass [27]. The antioxidant enzyme system (SOD, POD, and CAT) plays an important role in the protection of cells and organs from stress-related damage [28]. In the pot experiment, the straw of M. japonicus enhanced SOD, POD, and CAT activities of G. parviflora compared with that of the control, whereas the straws of R. sieboldii, C. confine, and P. asiatica reduced activities of these enzymes. This finding might be related to allelochemicals from M. japonica straw, which could enhance the tolerance of G. parviflora to Cd stress, whereas allelochemicals from R. sieboldii, C. confine, and P. asiatica straws might reduce the Cd stress tolerance of G. parviflora.
Under heavy metal contamination of the soil, studies on the effects of heavy metal absorption by plants have focused mainly on the rhizosphere environment [29][30]. Organic acids and other organic matter such as humus derived from the straw through decay and decomposition can change the rhizosphere pH, redox potential, and nutrient availability, and thereby affect the bioavailability of heavy metals in the rhizosphere soil [31][32]. In the current pot experiment, the soil pH was increased by the straws of R. sieboldii and C. confine, but decreased by the straws of M. japonicus and P. asiatica compared with that of the control. It may be that allelochemicals from R. sieboldii and C. confine straws promote the secretion of organic acids from the roots of G. parviflora during plant growth. In addition, in the pot experiment, the soil exchangeable Cd concentration was decreased by the straws of R. sieboldii and C. confine, and increased by the straws of M. japonicus and P. asiatica. The straws of R. sieboldii, C. confine, M. japonicus, and P. asiatica increased the Cd content in roots of G. parviflora compared with that of the control in the pot experiment, but only the straws of M. japonicus and P. asiatica increased the Cd content in shoots of G. parviflora in both the pot and field experiments. This result indicates that the organic acids and other organic matter derived from the straw by decay and decomposition can lead to increased Cd content in roots of G. parviflora, and only allelochemicals from M. japonica straw may increase the Cd content in shoots of G. parviflora. Thus, allelochemicals from different plant species may play different roles in Cd absorption and translocation by G. parviflora. The straws of M. japonicus and P. asiatica increased, whereas R. sieboldii and C. confine straws decreased, the amount of Cd extraction in roots of G. parviflora in the pot experiment compared with the control. Only M. japonicus straw increased the amount of Cd extraction in stems, leaves and shoots of G. parviflora compared with those of the control, whereas the other three straw types decreased Cd extraction in these organs, in both the pot and field experiments. Thus, the present results showed that the straw of M. japonicus can improve the phytoremediation ability of G. parviflora and can be used to improve the Cd extraction ability of G. parviflora from Cd-contaminated soil.