Effect of cadmium and arsenic contamination on element content and effectiveness in rhizosphere soils of different forages

To explore the effects of Cd and As composite contaminated soils on the element contents and effectiveness in rhizosphere soils of different forages, the root morphology, rhizosphere soil elements and their availability of 21 forages were studied and analyzed. The results showed: The 21 cultivars showed significant differences in root morphological characteristics. Root length is an index of root morphology that has the highest correlation with other elements and availability. And the correlation between the availability of each element and root morphology was higher than that of element content. Among them, Fe availability was significantly correlated with root morphological characteristics (P<0.05). The contents of different elements and availability varied greatly among the different cultivars. Among the six rhizosphere soil elements, the difference of toxic metal Cd was the largest, while the difference of Al availability was the largest. According to the correlation analysis between the elements and availability of each cultivar, the interaction of the availability of each element is higher than that of the soil element under the composite contaminated of Cd and As. Toxic metals Cd and As affected each other, and there was a very significant positive correlation between the content and availability. Fe, Mn and P had higher correlation with Cd and As.


Introduction
Toxic metal pollution is one of the important research problems in soil ecological remediation, which is shown not only by single element pollution, but also by two or even multi-element compound pollution [1] . Among them, the problem of cultivated land caused by Cd and As combined pollution is causing more and more economic losses to China. These two elements have a wide range of pollution and great toxicity. However, there are complex additions, synergism and antagonism between soil and organisms, which makes the impact of their combined pollution on the environment more complex than that of single pollution. The interaction between different metal elements also provides a new idea for biological detoxification and metal pollution remediation [2][3] . The interaction between metal and other elements is very complex under the stress of metal compound pollution, which will show different forms of action due to the existence of different elements. At present, the research on Cd and As compound polluted soil mainly focuses on the different absorption of toxic elements by cultivated varieties [4][5] . There are relatively few studies on the effects of metal pollution elements in soil on root morphology and the content of other elements in soil, which need to be studied.
Gejiu City, Yunnan Province is an industrial city rich in mineral resources. The problems left by the early mining and metallurgy are the main sources of Cd and As pollution in the local soil. According to the investigation, the local characteristic dairy industry has developed into a regular model, forages is the main forage crop. Therefore, 21 forages cultivars planted locally were used as test materials on the farmland soil polluted by Cd and As. The root morphological characteristics, different element contents and effective statuses of the forages cultivars were investigated to explore the interaction between each element under Cd and As composite contaminated soils. It is hoped to provide a basis for the future regulation of toxic metal elements in contaminated soils and the safe prevention and control during forages production in agricultural fields.

Experimental design
The test materials were sown in October 2019. In the insitu field test, each material was set up with three replicates. The plot area was 3m × 5m, and the materials were randomly distributed. In the in-situ field test, each material was set up with three groups of repetition, the plot area was 3m × 5m, and the materials were randomly distributed. During the growth period, insecticidal, weeding, watering and topdressing were carried out according to the growth status. Samples were collected in June 2020. Five sampling sites were sampled evenly and randomly by "X" method, and the plant root and rhizosphere soil samples were collected. The aboveground and underground parts of the plant were opened with scissors and put into polyethylene bags.

Determination of root morphology
The plant root scanner EPSON perfection V700 photo was used for scanning and analysis.

Determination of element content and available state in rhizosphere soil
The content of cadmium was determined by flame atomic absorption spectrometry (Thermo ICE 3000 SERIES) and recorded it in mg/kg [6][7] . The content of arsenic was determined by atomic fluorescence spectrometer (AFS-9710) and recorded it in mg/kg [8][9] . Determination of total phosphorus and available phosphorus in soil by molybdenum antimony anti colorimetric Spectrophoto-metry (Metash UV-5800) [6] . The content and effective state of Fe, Mn and Al were determined by ICP-OES (ThermoScientific iCAP6300) [6 、 10] .

Statistical analyses
The statistical significance analyzed used one-way analysis of variance (ANOVA) and Duncan test the difference of the average value of different treatments at the 0.05 level by SPSS 20.0 (n=3). And the correlation was analyzed by Spearman method.

Differences in rhizosphere soil pH and root morphology
The rhizosphere soil pH of the 21 cultivars ranged from 7.33 to 8.70, and the differences were not significant (P > 0.05), whereas obvious differences were observed in root morphology among the different cultivars (Table  2.). The maximum difference of total root length was 13.41 times, the maximum value was 'Denata' of Gramineae, and the minimum value was 'Grand slam' of Compositae, and the maximum differences of root length, root surface area, root volume and root diameter among cultivars were 1.97, 6.26, 30.82 and 179.3 times, respectively. Except for root length, the maximum value is Compositae. Note: Data are the mean ± standard errors of three replicates. Different lowercase letters represent significant differences between data in the same column (P < 0.05).

Difference of element content in rhizosphere soil
There were differences in the contents of different elements in the rhizosphere soil of different forage grasses (Figure 1.). The Cd and P contents in soil of 'Denata' was the highest. The highest contents of As, Fe, Mn and Al were 'You-12', 'Reyan 4', 'Faren' and 'Grand Slam', respectively, with significant difference (P < 0.05). After planting 'Reyan 4', the contents of Cd, As and Mn in rhizosphere soil were the lowest, the lowest contents of Fe were 'denata' and 'Dongmu-70', the lowest contents of Al were made in China, and the lowest contents of P were 'Munzhou I'. Among the six rhizosphere soil elements, the biggest difference was the Cd content, and the difference reached 4.76 times.

Difference of effective content of elements in rhizosphere soils
The highest availability of As and P was found in 'Denata' (Figure 2.), the lowest in 'Super King Tang' and '12SU9004', respectively. And 'Yemingzhu', 'Dongmu 70' and 'Super King Tang' had the highest availability of Cd, Mn and Al, while 'You-12', 'Reyan 4' and 'VNS' had the lowest. Moreover, The highest availability of Fe was 'Reyan 4', the lowest was 'denata' and 'Dongmu-70'. There were significant differences in rhizosphere soil availability among different cultivars, and the biggest difference among these available states was Al availa-bility, which was 4.22 times higher than that in rhizosphere soil of different cultivars.

Correlation analysis
The root morphology of forages was not significantly correlated with the content of Cd and As, the same as the soil pH (Table 3-5.). But the pH was significantly correlated with the As availability, and the root length was negatively correlated with the available state of Cd, Fe, Mn and P (P < 0.05). The contents of Cd and As were significantly positively correlated with the available state of Cd and As, and both of them were significantly correlated with Fe, Mn content and availability. (P < 0.05). There was extremely significant positive correl-ation between Cd, As availability and P availability (P＜0.01). The correlation of the available state of each element to the root morphology in the soil polluted by Cd and As was higher than that of the element content, and the interaction of the available state of each element was higher than that of the soil element.

Table 3. Correlation analysis between root morphology and soil elements among 21 cultivars.
Note: *At the 0.05 level, correlations were significant. ** At the 0.01 level the correlation was extremely significant. The same below.

Discussions
Roots showed a high degree of plasticity to changes of soil environmental factors during the growth process. Difference in genotype and growing environment can lead to changes in morphological characteristics of part or the whole root system [11] . Metal ions on the root surface can be absorbed actively and passively through cation exchange, diffusion and metabolic energy, and show a high selectivity to some metal elements [12] . Larger root morphological characteristics, such as root length, root surface area, root volume and root tip number, not only help to increase the contact area between plant roots and soil, and promote roots to absorb trace elements in soil. It may also increase the absorption and accumulation of metal elements [13] . In this experiment, the effect of pH on the rhizosphere soil of all forage cultivars before and after planting was small. There were significant differences in total root length, root surface area, root diameter and root volume among different forages, and the degree of difference varied with cultivars, which was consistent with the results of Gao et al. [14] . In root morphology, the total root length and root length of Gramineae were generally higher than those of Leguminosae and Compositae, while the root surface area, root diameter and root volume of Compositae were higher than those of Gramineae and Leguminosae, which was mainly related to the growth characters and interspecific differences of each material. Li et al. [15] also shows that the change in root form structure is an important embodiment of plant adaptation to stress. In addition, correlation analysis showed that there was a significant correlation between Fe availability and root morphology, and it was found that root length had more correlation with the content of Mn, P and the available state of Cd, Fe, Mn, which fully indicated that root length was a very important index in Cd and As contaminated soil, which may be more affected by some trace elements and Cd.
There are complex additive, synergistic and antagonistic effects between soil and organisms, which makes the impact of metal compound pollution on the environment more complex than that of single pollution [16] . Cd and As exist in different forms in soil with different migration characteristics and bioavailability. After Cd and As enter the soil environment, the interaction between soil pH, and hazardous metals is an important influencing factor [17] . Studies have shown that toxic elements tend to affect mineral elements in soils [18][19] . However, the contents of both toxic metals including Cd and As showed extremely significant positive correlation in this experiment (P < 0.01), and it indicated that the interaction had a large effect and showed a synergistic relationship. Guo et al. [20] reported that the coexistence of Cd and As in soil could additive to alfalfa, and Cd promote the accumulation of As. At the same time, the toxic elements were positively correlated with Fe content and negatively correlated with Mn content, but the effectiveness was positively correlated with the available state of Fe, Mn and P, which had obvious synergistic effect. This result suggests that increased or accumulated nutrients such as Fe and P in soils may exacerbate the risk of Cd or As contamination in cultivated forages.The interactive interaction between Cd, As and each element is an important factor affecting the absorption and accumulation of the pollutants toxic metals in soils by forages. It is similar to that of Cheng et al. [21] .

Conclusions
There were significant differences in root morphology among different cultivars, and the root length was more affected by some trace elements and Cd in the composite contaminated soils. The correlation between the available state of each element and root morphology was higher than that of element content, and the available state of rhizosphere soil elements had a greater effect on plant root morphology. There was a significant positive correlation between Cd and As, and the interaction was synergistic. At the same time, in the element content of rhizosphere soil, the difference of Cd content was the biggest, while in the available state, the difference of Al content was the biggest. The elements with high correlation with metal Cd and As are Fe, Mn and P. The results can provide a certain scientific basis for the selection of agricultural cultivars of farmland polluted by hazardous metals in mining area and the appropriate measures to control the pollution of Cd and As in farmland soil, and realize the effective control and safe utilization.