Effects of Pb on Rhizosphere Soil Enzyme Activity and Chemical Constituents of Achyranthes bidentata Blume

Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China

Abstract [Objectives] The purpose of this study was to investigate the effects of Pb on rhizosphere soil enzyme activity and chemical constituents of Achyranthes bidentata Blume. [Methods] A. bidentata Blume plants were cultivated in self-made polyvinyl chloride (PVC) pots. The soil was added with different levels of Pb (0, 200, 400, 600, 800 and 1 000 mg/kg air-dried soil) to investigate the effects of Pb on dry mass, active ingredients (oleanolic acid and ecdysterone) and rhizosphere soil enzyme activity of A. bidentata Blume. [Results] The root dry mass of A. bidentata Blume cultivated in the soil with Pb level above 400 mg/kg significantly reduced. The Pb residues in the A. bidentata Blume. plants growing in the soil with Pb level below 400 mg/kg complied with national standard. The contents of oleanolic acid and ecdysterone in A. bidentata Blume growing in the soil with Pb level above 600 mg/kg declined significantly. At the Pb level of 1 000 mg/kg, the activity of urease was inhibited significantly. The activity of phosphatase was inhibited in the presence of Pb in the soil. The activity of sucrase was activated in the soil with Pb level below 400 mg/kg, and was inhibited in the soil with Pb level above 400 mg/kg. [Conclusions] This study has important guiding significance for the reasonable selection of planting base for A. bidentata Blume and the guarantee of its yield and quality.

Key words Achyranthes bidentata Blume, Pb, Soil enzyme, Urease, Sucrase, Phosphatase

1 Introduction

AchyranthesbidentataBlume, Niuxi in Chinese, is a plant belonging to the family Polygonaceae, also known as Duijiecao. Its roots are generally used as a medicine (Radix Achyranthes Bidentata), with effects of nourishing liver and kidney, strengthening bones and muscles, removing blood stasis, stimulating menstrual flow, and guiding blood flow downward properly. As it is mainly produced in Jiaozuo, Henan (ancient Huaiqing Prefecture), it is also called Huainiuxi. It is one of the four famous medicines in Henan Province. Currently,A.bidentataBlume is mainly cultivated artificially. During the planting process, due to the exposure to Pb-contaminated irrigation water and air pollution from automobile exhaust, the soil in the planting area ofA.bidentataBlume is contaminated with Pb to varying degrees. At present, there have been many studies on the effects of Pb on field crops, but research on the effects of Pb onA.bidentataBlume has not been reported. Pb is not only harmful to plant growth[1], it can also damage the nervous, hematopoietic, digestive and immune systems of human body, causing greater harm to human body[2]. Excessive residue of Pb in Radix Achyranthes Bidentata will affect the medication safety and export of this medicinal material. In this study,A.bidentataBlume plants were cultivated in polyvinyl chloride (PVC) pots, and the soil was added with different levels of Pb to investigate the effects on Pb onA.bidentataBlume, so as to provide a theoretical basis for establishing the technical specifications ofA.bidentataBlume in conformity with national and international standards.

2 Materials and methods

2.1SoilandexperimentalplantsThe soil samples were collected from Jiabu 1 Village, Wuzhi County, Henan Province, a typical producing area ofA.bidentataBlume. The type of the soil was calcareous fluvo-aquic soil developed from the Yellow River alluvium, and the texture was light soil. The organic matter, total N, total P, total K and total Pb contents and pH value of the soil were 10.8 g/kg, 1 670.6 mg/kg, 1 640.4 mg/kg, 950.2 mg/kg, 72 mg/kg and 7.85 (soil∶water = 1 g∶10 mL), respectively. The test cultivar ofA.bidentataBlume was Fengzhengke.

2.2ExperimentdesignSelf-made PVC pipes (1.2 m in length and 12 cm in diameter) were used. They were buried in the ground, and the upper ends were 5 cm higher than the ground. Iron sheet was bent into cylinders (1.2 m in length and 6 cm in diameter), which were then jacketed with 300-mesh nylon mesh and placed into the PVC pipes. Soil was poured into the PVC pipes, inside and outside the iron sheet. Then, the iron sheet was pulled out. When filling soil, the lower part of each PVC pipe (0-80 cm) was filled with 12.5 kg of soil, and the upper part of each PVC pipe (80-95 cm) was filled with 2.0 kg of soil mixed with lead acetate. When preparing mixed soil, corresponding amounts (0, 200, 400, 600, 800 and 1 000 mg/kg air-dried soil) of lead acetate were added to the soil, respectively according to the experimental design. In the soil in the upper part of each PVC pipe, 200 mg of P (in the form of potassium dihydrogen phosphate), 240 mg of N (in the form of urea) and 200 mg of K (in the form of potassium sulphate) were added. Four replicates were arranged for each treatment. The sedimentation was carried out for 10 d.

On July 6, 2015, 5 seeds ofA.bidentataBlume were directly sown at the center of each PVC pipe. After half a month, one plant was retained for each PVC pipe. During the growth period, distilled water was irrigated according to the soil moisture. Harvest was carried out in early to mid-November. The soil inside the nylon mesh was the rhizosphere soil, and the soil outside the nylon mesh was the non-rhizosphere soil. All the soil samples were collected from the upper part of the PVC pipes. The above-ground and under-ground parts of the plants were harvested separately. The roots of each plant were rinsed with distilled water, dried at 70 ℃ and weighed.

2.3DeterminationmethodsThe activity of soil enzymes was measured referring to the methods of Zheng Hongyuan[3]. The activity of sucrase was determined using the dinitrosalicylic acid colorimetric method, and it was expressed as glucose content in 1 g of soil after 24 h (mg/g). The activity of urease was determined using indophenol blue colorimetry, and it was expressed as the content of NH3-N in 1 g of soil after 24 h (mg/g). The activity of phosphatase was determined with disodium phenyl phosphate colorimetry, and it was expressed as the content of phenol released in 1 g of soil after 24 h (mg/g).

The contents of oleanolic acid and ecdysterone inA.bidentataBlume and the content of Pb in Radix Achyranthes Bidentata were determined according to the 2015 edition of thePharmacopoeiaofthePeople’sRepublicofChina(hereinafter referred to asPharmacopoeia)[4].

2.4DataanalysisData statistics was performed using one-way ANOVA of SPSS 20.0.

3 Results and analysis

3.1EffectofPbonqualityofA.bidentataBlumeAs shown in Table 1, compared with the control group (Pb level of 0 mg/kg), the root dry mass ofA.bidentataBlume growing in the soil with Pb level of 200 mg/kg increased slightly (P>0.05), and the root dry mass ofA.bidentataBlume growing in the soil with Pb level equal to and above 400 mg/kg reduced significantly (P<0.05). It indicates that Pb is not an essential element for the growth ofA.bidentataBlume; in the case of low residual level, it has little effect on the growth of roots ofA.bidentataBlume; but when the Pb level is equal to and higher than 400 mg/kg, it has a toxic effect on the growth of roots ofA.bidentataBlume, adversely affecting the growth ofA.bidentataBlume roots.

The content of Pb in Radix Achyranthes Bidentata increased continuously with its increased residual level in the soil. Compared with that of the control group, the contents of Pb in Radix Achyranthes Bidentata of the treatment groups changed significantly (P<0.05). It suggests that excessive residue of Pb in the soil is one of the key factors for the excessive residue of Pb in the medicinal materials. ThePharmacopoeia(2015 edition) stipulates that the residual level of Pb in medicinal materials should not exceed 5.0 mg/kg[4]. Under the soil Pb level of 400 mg/kg, the Pb residue in Radix Achyranthes Bidentata was close to the maximum limit stipulated by thePharmacopoeia. When the soil residual level was equal to and lower than 200 mg/kg, the Pb residue in RadixAchyranthesBidentatamet the requirement. Considering the residual level of Pb in Radix Achyranthes Bidentata, the Pb level in the soil for growingA.bidentataBlume should be below 250 mg/kg.

Table1EffectsofPbinthesoilonrootdrymassandPbresidueofAchyranthesbidentataBlume

Pb level∥mg/kgRoot dry mass∥g/plantPb residue∥mg/kg019.34±0.040.890±0.01220019.38±0.033.587±0.022∗10019.12±0.03∗5.233±0.149∗60016.36±0.03∗7.385±0.076∗80011.98±0.03∗8.427±0.081∗1 0009.67±0.05∗9.665±0.061∗

Note: Analysis was performed using F-test; compared with the control group (Pb level of 0 mg/kg),*P<0.05. The same in Table 2.

3.2EffectsofPbonactiveingredientsinA.bidentataBlumeAs shown in Table 2, when the soil Pb level was not higher than 400 mg/kg, the contents of oleanolic acid and ecdysterone inA.bidentataBlume showed no significant differences compared with those of the control group (P>0.05). When the soil Pb level was equal to and higher than 600 mg/kg, the contents of oleanolic acid and ecdysterone inA.bidentataBlume declined significantly compared with those of the control group (P<0.05). Wang Xinmin studied the effects of Cu on active ingredients of Radix Achyranthes Bidentata, and the results show that the contents of oleanolic acid and ecdysterone in Radix Achyranthes Bidentata still not changed significantly when the soil Cu level reached 600 mg/kg[5], different from the results of this study.

Table2EffectsofPbinthesoilonoleanolicacidandecdysteronecontentsofAchyranthesbidentataBlume

Pb level∥mg/kgOleanolic acid∥%Ecdysterone∥%01.06±0.010.15±0.012001.06±0.010.14±0.014001.04±0.010.13±0.026000.95±0.01∗0.11±0.01∗8000.87±0.02∗0.09±0.01∗1 0000.65±0.03∗0.07±.01∗

3.3EffectsofPbonactivityofenzymesinrhizospheresoilofA.bidentataBlumeAs shown in Fig.1, when the soil Pb level was below 800 mg/kg, the activity of urease was not affected; and when the soil Pb level reached 1 000 mg/kg, the activity of urease was significantly inhibited compared with the control group, similar to the findings of Xie Siqin on effects of heavy metals on soil enzyme activity[6]. As shown in Fig.2, the activity of sucrase inA.bidentataBlume growing in the soil with Pb level equal to and below 400 mg/kg was enhanced, and that inA.bidentataBlume growing in the soil with Pb level higher than 400 mg/kg was inhibited compared with the control group. As shown in Fig.3, the activity of phosphatase inA.bidentataBlume declined gradually as the soil Pb level increased, consistent with the research result of Zhou Furongetal[7]. The reason for enhancing effect of low-level Pb and inhibitory effect of high-level Pb on sucrase may be that the acetate ions in lead acetate increased the carbon in the soil and promoted the growth of microorganisms, leading to increased sucrase activity in the soil, and thus, the damage of low-level Pb on enzyme activity was not shown[8]. However, the damage caused by high-level Pb on soil sucrase could not be compensated by the role of acetate ions. As a result, when the soil Pb level exceeded 600 mg/kg, the activity of sucrase declined; and the higher the soil Pb level, the more obvious the decrease in sucrase activity.

Fig.1EffectsofPbontheactivityofurease

Fig.2EffectsofPbontheactivityofsucrase

Fig.3EffectsofPbontheactivityofphosphatase

4 Conclusions

Low level of Pb in soil will not produce a significant impact on the yield of Radix Achyranthes Bidentata, and its effects on the contents of oleanolic acid and ecdysterone in Radix Achyranthes Bidentata are little. However, the Pb residue in Radix Achyranthes Bidentata is increased. Excessive Pb in the soil will not only reduce the yield of Radix Achyranthes Bidentata but also reduce the contents of active ingredients and increase the residue of Pb in Radix Achyranthes Bidentata. In this study, high-level Pb reduced the activity of most enzymes in the soil. Soil enzyme activity is used to characterize the degree of maturity and fertility of soil. Pb affected the absorption of mineral elements by the root system ofA.bidentataBlume by changing the activity of enzymes in the soil, thus affecting the entire physiological activity ofA.bidentataBlume. The effects of Pb on soil enzyme activity and dry root mass ofA.bidentataBlume were basically the same in this study. When plantingA.bidentataBlume, the Pb content in the soil should be detected first, and the soil with Pb content in a safe range is recommended to ensure the yield and quality of Radix Achyranthes Bidentata.