Improving soil selenium availability as a strategy to promote selenium uptake by high-Se rice cultivar

Accepted Manuscript Title: Improving soil selenium availability as a strategy to promote selenium uptake by high-Se rice cultivar Authors: Mu Zhang, Guofang Xing, Shuanhu Tang, Yuwan Pang, Qiong Yi, Qiaoyi Huang, Xu Huang, Jianfeng Huang, Ping Li, Hongting Fu PII: DOI: Reference:

S0098-8472(19)30154-6 https://doi.org/10.1016/j.envexpbot.2019.04.008 EEB 3752

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

31 January 2019 8 March 2019 8 April 2019

Please cite this article as: Zhang M, Xing G, Tang S, Pang Y, Yi Q, Huang Q, Huang X, Huang J, Li P, Fu H, Improving soil selenium availability as a strategy to promote selenium uptake by high-Se rice cultivar, Environmental and Experimental Botany (2019), https://doi.org/10.1016/j.envexpbot.2019.04.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Improving soil selenium availability as a strategy to promote selenium uptake by high-Se rice cultivar Author names Mu Zhang a, b, Guofang Xing c, Shuanhu Tang* a, b, Yuwan Pang a, b, Qiong Yia,b , Qiaoyi

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Huanga, b, Xu Huang a, b, Jianfeng Huang a, b, Ping Lia, b, Hongting Fua, b

Affiliations a

Institute of Agricultural Resources and Environment, Guangdong Academy of Agricultural

b

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Sciences, Guangzhou 510640, China

Key Laboratory of Plant Nutrition and Fertiliser in South Region, Ministry of Agriculture,

Guangzhou 510640, China

College of Agronomy, Shanxi Agricultural University, Taigu 030801,China

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c

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*Corresponding author

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Shuanhu Tang; E-mail address: [email protected].

Highlights

The grain Se contents of the high-Se rice cultivar were significantly higher than

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those of the low-Se rice cultivar in Se treatments. The high-Se rice cultivar could obtain more Se from non-rhizosphere soil than the low-Se

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rice cultivar.

Both of the organic acid secretions of the two rice cultivars decreased at the high

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Se level.

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The infrared absorption intensities of rhizosphere soils of the two rice cultivars were increased by the application of Se, while the high-Se rice cultivar exhibited a greater degree of increase than the low-Se rice cultivar.

Abstract The aim of this research was to investigate the mechanism of selenium (Se) utilization by

roots between two rice genotypes. The plant Se concentration, root organic acids secretion, soil Se fractions, soil pH, and infrared spectrum of soil were all determined through a root box experiment to clarify the differences in Se availability of rhizosphere soils between high- and low-Se rice cultivars. The results revealed that the grain Se concentration of the high-Se cultivar was significantly higher than that of the low-Se cultivar. The concentration of available Se, e.g. water-soluble and exchangeable, in the rhizosphere soil of the high-Se rice cultivar was dramatically higher than that of the low-Se rice cultivar, and the opposite result

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was observed in non-rhizosphere soil. The organic acids secretion of the high-Se rice cultivar showed a greater degree of reduction than that of the low-Se rice cultivar in response to the

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increase of Se application rates, while the infrared spectrum intensity of clay mineral in the

rhizosphere of the high-Se rice cultivar exhibited a greater degree of increase than that of the low-Se rice cultivar. In addition, the pH of the rhizosphere soil of the high-Se rice cultivar was significantly higher than that of the low-Se rice cultivar at 0 mg kg-1 Se treatment. The

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results demonstrated that the high-Se rice cultivar could obtain more Se than the low-Se rice

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cultivar by mass flow, which is limited by the concentration of mobile Se in soil; The high-Se rice cultivar had a greater ability to activate Se by increasing soil pH than the low-Se rice

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cultivar; The secretion of organic acids could activate Se by degrading clay minerals, while

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the high-Se rice cultivar may have a greater ability to regulate the secretion of organic acids. The present study suggests the high-Se rice cultivar has stronger ability to increase Se

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availability of rhizosphere soil than the low-Se rice cultivar.

Keywords: Rice; selenium; availability; rhizosphere; organic acid

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1 Introduction

Rice (Oryza sativa L.) is a major food crop in Asia (Seck et al., 2012), and occupies a

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worldwide area of at least 160 million ha; thus, it is the preferred crop to use for fortification with selenium (Se) in Asia. However, studies have shown that grain Se concentration varied significantly between rice cultivars grown in soil with the same Se level (Lidon et al., 2018; White, 2017). The theory of mineral nutrition suggests that differences in Se concentration in

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grains occur as a result of different transport and absorption mechanisms, and that the processes are mainly due to the plant genotype (Marschner, 2012). Therefore, selective breeding would be convenient if rice crops possess significant differences in grain Se concentrations. The method is practical, but does not, however, explain why the grains of rice cultivars grown in areas exposed to the same Se levels differ in Se accumulation. Selenium is primarily adsorbed from the soil by plants as selenite (SeO32-) or selenate

(SeO42-) , which are two common oxidation species in soil. Selenite is tightly bound to positively charged binding sites in the soil, and thus only little available and mobility for plant uptake (Eich-Greatorex et al., 2010; Peng et al., 2016). The uptake efficiency of selenite by plant roots is markedly lower than that of selenate (Seppänen et al. 2010), and also the translocation of selenite from roots to shoots occurs less readily than selenate (Sors et al., 2005). Previous studies have shown that rice mostly uptakes selenite, which is the dominant speciation of Se in waterlogged paddy soils due to the low redox potential (Diyabalanage et

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al., 2016). Therefore, the amount of Se absorbed by rice mainly depends on the absorption capacity of selenite. Although no available evidence demonstrates that the plants exhibit

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specificity in Se transporters, several studies have verified that selenite is transported via

phosphate transporters (Zhang et al., 2014). The relative expression levels of the transporter genes are related to plant genotypes (White, 2015), and affect the efficiency of absorption of soil Se. Consequently, the effect caused by genotypic differences will eventually be reflected

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in the absorption of the rhizosphere soil Se. Therefore, the variation characteristics in the

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rhizosphere and non-rhizosphere soil Se contents may provide some clues for the study of

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different Se absorption mechanisms among different rice cultivars. When Se is added to soil, it undergoes physical changes and chemical reactions with soil

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components and it will be distributed in soil components. Thus, the bioavailability and mobility of Se not only depend on the speciation and total Se concentration but also more

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important on Se fractions (Li et al., 2016). To better assess the soil Se availability, the chemical association of Se with other soil components needs to be further clarified.

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Extractable Se in rhizosphere soil generally provides a better indication of Se availability in rice (Fan et al., 2015). A sequential extraction procedure was proposed by Li et al. (2016), who showed that soil Se is operationally fractionated into water-soluble, exchangeable, Fe-

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Mn oxide-bound, organic matter/sulphide-bound and residual fractions. The bioavailability and mobility of Se in soil strongly depends on its fraction in the soil (Sharma et al., 2015). The plant-available Se in the soil, such as water-soluble and exchangeable Se, consists of

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mobile fractions that are readily taken up by plants. Plants are the main driving force for nutrient movement from non-rhizosphere to rhizosphere soil (Chapin lll et al., 2011). Therefore, the movement efficiency of a nutrient from non-rhizosphere to rhizosphere soil may be considered to reflect the potential capability of the plants to uptake nutrients. Although many studies have investigated the Se absorption capacity of rice cultivars in relation to the fraction and availability of Se in soils, the difference in Se movement from non-rhizosphere to rhizosphere soil between high- and low-Se rice cultivars is not yet well

understood. The root is the major plant organ responsible for the uptake of nutrients and the release of organic substances; thus, the root may affect the bioavailability of Se in soil, particularly in rhizosphere soil. Previous studies have shown that plant roots exude many organic compounds, including organic acids, amino acids, proteins, phenolics and secondary metabolites (Erb et al., 2013; Huang et al., 2014). Among the many organic substances,

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organic acids are closely related to the availability of soil nutrients (Maseko and Dakora, 2013; Rengel, 2015; Dotaniya et al., 2015). To the best of our knowledge, many studies have primarily demonstrated that plants could activate P, Fe, Mo and Zn in soils by root exudates

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(Maqsood et al., 2011; López-Millán et al., 2012; Zhao and Wu, 2014), but little attention has been paid to the relationship between soil Se availability and organic acid secretion. It is presently unclear whether the secretion of organic acids from rice will affect the availability of soil Se. Information on soil composition will also help to provide a more comprehensive

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understanding of the relationship between organic acid secretion and soil Se availability.

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Studies have reported that various soils have their characteristic infrared absorption spectrum,

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related to the soil character (Du et al., 2009; Vohland et al., 2014). In our experiment, the

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absorption spectrum of the soil was proven to be that of a kaolinite-type clay mineral. To further elaborate the mechanism of the uptake of Se by rice, high- and low-Se rice cultivars were selected for testing. We hypothesized that Se uptake is affected by the

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concentration of available Se in rhizosphere soil and by the movement of Se from nonrhizosphere to rhizosphere soil, secondly, that the soil Se fraction affects Se availability and

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mobility, and finally, that rice may be one of the active driving forces for improving Se bioavailability and mobility. The aims of the present study are (1) to investigate the effects of

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Se on root, leaf and grain Se concentrations, (2) to determine the fractions of Se in rhizosphere and non-rhizosphere soil, (3) to measure the secretion of organic acids and the infrared absorption spectrum intensity of rhizosphere soil, and (4) to elucidate the relationship between the Se absorption capacity of rice and the available Se concentration, Se fraction,

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organic acid secretion and infrared absorption spectrum of rhizosphere soil.

2 Materials and methods 2.1 Materials and experimental treatments A rhizobox experiment was performed at the Guangdong Academy of Agricultural Sciences, China (23°7′ N, 113°15′ E). The meteorological conditions of the greenhouse were as follows: 24/32 °C night/day temperature, 75% relative humidity and natural light. The chemical

properties of the experimental soil were as follows: pH 5.0, organic matter 41.1 g kg-1, alkaline hydrolysable N 168.6 mg kg-1, Olsen-P 18.7 mg kg-1, available K 148.5 mg kg-1, available Fe 55.2 mg kg-1, available Mn 11.6 mg kg-1, available Cu 2.3 mg kg-1, available Zn 4.3 mg kg-1, available B 0.40 mg kg-1, total Se 0.34 mg kg-1 and available Se 0.06 mg kg-1. The experimental soil was air-dried and sieved to a 20-mesh powder. Each rhizobox was filled with 2 kg of soil and fertilized with the following macroelements (g kg-1 soil): 0.2 N, 0.15 P2O5 and 0.2 K2O, supplied in the form of CO(NH2)2, (NH4)2HPO4 and KCl, respectively. The

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microelements supplied were 0.08 mg of CuSO4·5H2O, 1.81 mg of MnCl2·4H2O, 2.86 mg of H3BO3 and 0.22 mg of ZnSO4·7H2O per kg of soil. Treatments included three concentrations

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of Se (0, 0.5 and 5.0 mg kg-1 soil), and Na2SeO3 of analytical grade was used as Se fertilizer. All of the fertilizers were dissolved in water and then applied to the soil before sowing. The rhizobox (15-cm length, 10-cm width, 16-cm height) contained two outer chambers (4-cm width) and one inner chamber (2-cm width) separated by a membrane with a pore size of 30

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μm (Figure 1). The inner chamber contained rhizosphere soil, and the outer chamber

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contained non-rhizosphere soil (Li et al., 2000). In this experiment, Oryza sativa L. low grain Se cultivar Hefengzhan and high grain Se cultivar Fengbazhan were used as test materials.

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The experimental layout was a randomized block design containing eight replicates per

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treatment, and seedlings were thinned to one per box. 2.2 Analysis of soil chemical properties

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The soil chemical properties were determined by the methods of Bao (2002). The pH (soil water ratio of 1:5) was determined by the PB-10 pH meter (Sartorius, Germany). The organic

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matter was determined by using volumetric method with the potassium dichromate. The alkaline hydrolysable N was measured by the method of alkali dissociation diffusion. The Olsen-P was extracted by 0.5 M NaHCO3, and the concentration of P was determined by

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using colorimetric method with the molybdenum-antimony. The available K was extracted by 1.0 M NH4OAc, and the extract was analyzed by using a FP6410 flame photometer (Xingyi, China). The available Fe, Mn, Cu and Zn were extracted by 0.005 M DTPA-0.01 M CaCl2,

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and the concentration of micronutrient in the extract was determined by using a ZA3300 Atomic Absorption Spectrophotometer (Hitachi, Japan). The available B was extracted by the method of hot water reflux leaching, and the extract was measured by using colorimetric method with the curcumin. The soil available Se was calculated by the sum of soluble and exchangeable Se, and the procedure was presented in the part of Se fractions analysis. For soil total Se analysis, 1.0 g of soil was digested with 6 mL ultra-pure nitric acid and 2 mL

perchloric acid at 160℃ until the soil turned gray. 10 ml of 6 M hydrochloric acid was then added and the digestion was completed by heating at 100℃ for 10min. Concentration of Se in the solution was analyzed by using an 8200 Atomic Fluorescence Spectrometer (Jitian, China) (NY/T, 2006). The certified reference material (Soil, GBW07408/GSS-8) was used as the quality control sample to calculate the recovery rate of soil Se (94.9 %). 2.3 Analysis of plant Se The rice samples for each treatment were collected at harvest, washed several times with

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deionised water and then oven-dried at 55 ºC. The oven-dried samples were ground and

filtered using a 1-mm nylon sieve for nutrient analysis. For the Se assays, the rice samples

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were digested in 8 mL of ultra-pure nitric acid and 2 mL of perchloric acid at 170 ºC. The acid mixture was heated until white smoke appeared, and 10 mL of 6 M hydrochloric acid was then added. The concentration of Se in the solution was determined using an 8200 atomic fluorescence spectrometer (JITIAN Company, China) (GB/T, 2008). The certified reference

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material (Bush Twigs and Leaves, GBW07603/GSV-2) was used as the quality control sample

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to calculate the recovery rate of Se (96.5%).

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2.4 Analysis of rhizosphere and non- rhizosphere soil Se fractions The Se fractions were extracted according to the sequential extraction (Qu et al., 1997; Liu et

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al., 2015). In brief, dry 1.0 g of soil sample was weighed into a 50 ml centrifuge tube. The procedure of the sequential extraction was as follows:

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Soluble Se: 10 ml of 0.25 M HCl was added to the centrifuge tube and shaken at 200 rpm at 25 ºC for 1 h. The homogenate was centrifuged at 4000 rpm for 10 min, then the supernatant

analysis.

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was collected. The extraction was repeated twice, and the supernatants were used for Se

Exchangeable Se: 10 ml of 0.7 M KH2PO4 was added to the previous residue and shaken at

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200 rpm at 25 ºC for 4 h, then centrifuge at 4000 rpm for 10 min. Fe/Mn oxide-bound Se: 10 ml of 2.5 M HCl was added to the previous residue, and the mixture was heated in a water bath at 90 ºC for 50 min with intermittent shaking, then

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centrifuge at 4000 rpm for 10 min. Organic matter/sulfide-bound Se: 8 ml of 5 % K2S2O8 and 2 ml of 1:1 HNO3 were added to the previous residue, and the mixture was heated in a water bath at 95 ºC for 3 h with intermittent shaking, then centrifuge at 4000 rpm for 10 min. Residual Se: The soil residue was digested in 10 ml of HNO3-HClO4 (3:2) at 160 ºC by using the method of soil total Se analysis. Concentration of Se in the extraction solution was analyzed by using an 8200 Atomic

Fluorescence Spectrometer (Jitian, China). The certified reference material (Soil, GBW07408/GSS-8) was used as the quality control sample to calculate the recovery rate of the sum of Se concentrations in various fractions (108.7%). 2.5 Analysis of rhizosphere organic acid Twenty-five days after sowing, four replicate seedlings were carefully washed, disinfected with 0.03 mM thymol and transplanted to brown bottles with 50 ml of 5 mM CaCl2. Root exudates were collected over a period of 12 h in a light-entrainment room. Consumption of

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the CaCl2 solution was measured by monitoring the weight and then adding the correct

amount of deionized water. The solution was filtered and then diluted in 50 mL of ultra-pure

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water. The solution was freeze-dried and stored at -20 ºC for organic acid analysis (Zhao et al. 2013). The freeze-dried samples were dissolved in 10 mL of ultra-pure water and filtered through 0.45 μm Millipore filters prior to the organic acid analysis by HPLC (Waters, USA). The analytical column was a Waters C18 column (4.6 mm × 250 mm, 5 μm), and the mobile

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phase for the HPLC was 10 mM (NH4)2HPO4 (pH 2.7, 1.0 mL min-1). The detector used for

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the HPLC analysis was an ultraviolet absorption detector (UVD). The standard solution was prepared as follows: oxalic acid, malic acid, malonic acid, acetic acid and tartaric acid

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(Sigma, USA) dissolved in ultra-pure water. The standard curve range was 0 to100 mg/L

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(Zhao et al., 2013).

2.6 Analysis of rhizosphere soil infrared absorption spectrum

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Rhizosphere soils from each treatment were collected for an infrared absorption spectrum analysis. The samples were dried under normal atmospheric temperature and then mixed in

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equal amounts to form four replicates for each treatment. The infrared absorption spectrum of the soils was measured using a potassium bromide tablet and a Tensor 27 Fourier Transform Infrared Spectrometer (Bruker Optics, Germany).

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2.7 Data analysis

All the data were statistically analyzed using SPSS 12.0 software, and the mean values of each treatment group were subjected to a multiple comparisons analysis by the LSD-test with

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a significance level of p<0.05. All figures were drawn using Sigma Plot 10.0 software.

3 Results 3.1 Effect of Se on the grain yield and fresh weight in the two rice cultivars. Figure 2 displays the grain yields and fresh weight of two rice cultivars, cultivated in the presence of different concentrations of Se. Grain yields of the two rice cultivars were both significantly increased (p<0.05) due to the application of Se (Figure 2a), and no significant

difference was found between the two rice cultivars. The application of Se significantly increased the fresh weight of the high-Se (H-Se) rice cultivar, but there was no dramatic effect on the fresh weight of the low-Se (L-Se) rice cultivar (Figure 2b). 3.2 Effect of Se on the root, shoot and rice grain Se contents at maturity in the two rice cultivars. The rice grain Se concentrations of the two rice cultivars were both significantly increased by

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the application of Se, and the highest grain Se concentrations of the two rice cultivars were

both found in the area treated with 5.0 mg Se kg-1 (Figure 3a). A comparison of the grain Se

concentrations of the two rice cultivars exposed to the same Se application treatments showed

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that the grain Se content of the high-Se rice cultivar was significantly higher than that of the

low-Se rice cultivar. The root and shoot Se concentrations of the two rice cultivars were also significantly increased by the application of Se, and were both positively correlated with the

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concentration of Se applied (Figure 3b; 3c). The shoot and root Se concentrations of the highSe rice cultivar were both significantly higher than those of the low-Se rice cultivar in the 5.0

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mg Se kg-1 treatment, but there was no dramatic differences in the 0 and 0.5 mg Se kg-1

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treatments.

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3.3 Effect of Se on the organic acid secretions in the two rice cultivars. As shown in Figure 4a, the total organic acid secretions of the two rice cultivars were both

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significantly decreased by the application of Se. There were no significant differences in total organic acid secretions between the two rice cultivars in the 0 and 0.5 mg kg-1 Se levels, but

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the total organic acid secretion of the high-Se rice cultivar was dramatically lower than that of the low-rice cultivar at the 5.0 mg kg-1 Se level. The application of 0.5 and 5.0 mg kg-1 Se both significantly decreased oxalic acid, malic acid, acetic acid, malonic acid, and tartaric

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acid secretions of the low-Se rice cultivar (Figure 4b; c; d; e; f). However, the response in organic acid secretion of the high-Se rice cultivar to Se varied; the application of 0.5 mg kg-1 Se increased oxalic acid, malic acid, and malonic acid secretions, and the application of 5.0

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mg kg-1 Se dramatically decreased oxalic acid, malic acid, acetic acid, malonic acid, and tartaric acid secretions. Oxalic acid, malic acid, acetic acid, and malonic acid secretions of the high-Se rice cultivar were significantly higher than those of the low-Se rice cultivar at the 0.5 mg kg-1 Se level, and the tartaric acid secretion of the high-Se rice cultivar was dramatically lower than that of the low-Se rice cultivar. However, Oxalic acid, malic acid, malonic acid and tartaric acid secretions of the high-Se rice cultivar were notably lower than those of the low-rice cultivar at the 5.0 mg kg-1 Se level.

3.4 Effect of Se on the rhizosphere and non-rhizosphere soil pH of the two rice cultivars. The pH values of the rhizosphere soils were higher than those of the non-rhizosphere soils in the two rice cultivars (Figure 5a; b). The pH of the rhizosphere soils of the two rice cultivars were both remarkably decreased due to the application of Se, and the lowest pH values both occurred in the 5.0 mg Se kg-1 treatment. The pH of the rhizosphere soil of the high-Se

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cultivar was significantly higher than that of the low-Se cultivar at the 0 mg Se kg-1 treatment, but there were no significant differences in the pH of the rhizosphere soils between the two rice cultivars in the 0.5 and 5.0 mg Se kg-1 treatments. The application of Se had no

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significant effects on the pH of the non-rhizosphere soils in both of the rice cultivars.

3.5 Effect of Se on the various fractions of Se concentrations and proportions in the

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rhizosphere and non-rhizosphere soils of the two rice cultivars.

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The total Se concentrations in rhizosphere and non-rhizosphere soils of the two rice cultivars

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were both significantly increased by the application of Se, and the highest Se concentrations were found in the area treated with 5.0 mg Se kg-1 (Figure 6). The rhizosphere soil Se

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concentration of the high-Se cultivar was significantly higher than that of the low-Se cultivar in the 5.0 mg Se kg-1 treatment (Figure 6a), but the Se concentration in the non-rhizosphere

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soil displayed the opposite (Figure 6b). There were no significant differences in the rhizosphere and non-rhizosphere Se concentrations between the two rice cultivars in the 0 and

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0.5 mg Se kg-1 treatments.

The water-soluble, exchangeable, Fe/Mn oxide-bound, organic matter/sulphide-bound and

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residual Se concentrations in the rhizosphere and non-rhizosphere soils of the two rice cultivars were all significantly increased by the application of Se (Figure 7), and the highest values all occurred in the area treated with 5 mg Se kg-1 soil. There were no significant differences in the water-soluble, exchangeable, Fe/Mn oxide-bound, organic matter/sulphide-

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bound and residual Se concentrations of the rhizosphere and non-rhizosphere soils between the high- and low-Se rice cultivars at the 0 and 0.5 mg Se kg-1 treatments. The water-soluble, exchangeable, Fe/Mn oxide-bound, organic matter/sulphide-bound Se concentrations in the rhizosphere soil of the high-Se rice cultivar were significantly higher than those of the low-Se rice cultivar in the 5.0 mg kg-1 Se level, but the water-soluble, exchangeable, Fe/Mn oxidebound and residual Se concentrations in the non-rhizosphere of the high-Se rice cultivar were

dramatically lower than those of the low-Se rice cultivar at the same Se level. The proportions of Se in various fractions are shown in Figure 8; the line of proportion went from the organic matter/sulphide bound to the residual and exchangeable and then down to the Fe/Mn oxide-bound, and finally to water-soluble in the rhizosphere and non-rhizosphere soils. Soil Se occurred primarily in the organic matter/sulphide-bound, which accounted for nearly 60% of the total Se. There was no significant difference in the proportion of various

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fractions of Se between the two rice cultivars. 3.6 Effect of Se on the infrared absorption spectrum of the rhizosphere soils of the

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two rice cultivars.

Figure 9 shows the infrared absorption spectrum of rhizosphere soil of the two rice cultivars, cultivated with various concentrations of Se. The infrared absorption intensity（3697.21, 3621.17 and 1031.95 cm-1）of the rhizosphere soil in which the high-Se rice cultivar was

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cultivated was increased by the application of Se. Furthermore, with the addition of Se (>0.5

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mg kg-1), the infrared absorption intensity of the rhizosphere soil of the high-Se rice cultivar was significantly higher than that of the low-Se rice cultivar. The results indicated that the

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kaolinite-type clay mineral concentration in the rhizosphere soil of the high-Se rice cultivar

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was higher than that of the low-Se rice cultivar exposed to an appropriate concentration of Se.

and plant items.

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3.7 Pearson correlation coefficients for the relationship between the rhizosphere soil

Statistical analysis showed that there were significant correlations between soil and plant

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items in this experiment (Table 1). The concentrations of Se in grains, leaves and roots of the two rice cultivars were all positively related to the Se concentrations in the different soil Se

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fractions. However, the concentrations of oxalic acid, tartaric acid, malic acid, malonic acid, and acetic acid were all negatively correlated to the Se concentrations in the different rhizosphere soil Se fractions. The pH of the rhizosphere soil was negatively correlated with the Se concentration of plant tissues, and positively correlated with the secretion of organic

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acids.

4 Discussion Although no rice cultivars are Se hyperaccumulators, rice cultivars can be differentiated into high-Se and low-Se cultivars (Banuelos et al., 2013; Reis et al., 2013). Previous studies focused on measuring the grain Se contents and related yields, with results demonstration substantial differences in the grain Se contents of different rice cultivars, exposed to the same

soil Se concentration (Reis et al., 2015; Gong et al., 2018). The present study also showed that the difference in grain Se accumulation between the two rice cultivars originates from genotypic differences, as was determined after removal of the dilution effect caused by different grain yields. Previous reports have demonstrated that the difference in grain Se contents between numerous rice cultivars depends on the root absorption capacity and xylem transport capacity (Lu et al., 2013; Wu et al., 2015). The Se concentrations in grains of the high-Se rice cultivars were significantly higher than those of the low-Se rice cultivar in the

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0.5 and 5.0 mg Se kg-1 treatments.

Although the processes of transport and absorption are mainly determined by the genotype of

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rice, results showed that the two processes are also limited by the bioavailability of Se in

rhizosphere soil. The results showed that in soils with applied Se, the Se occurred primarily in the organic matter/sulfide-bound and residual fractions, whereas the rice available fractions of Se only occupied a minor proportion of the total Se in soil, comprising the water-soluble and

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exchangeable Se. A previous study showed that selenite is strongly absorbed by soil minerals

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and thus has a low bioavailability in acidic soil (Stroud et al., 2010). As selenite was applied

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in this experiment, this is a primary reason for the detection of a low concentration of

the mobility of Se in this experiment.

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available Se in the Se treatments. Furthermore, the immobilization of Se in soil also decreased

The movement of nutrients through the soil system to the plant root surface occur via two

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pathways (Oliveira et al., 2010, Oyewole et al., 2014): the movement of Se along with water that is absorbed by the root, and the movement of Se due to the nutrient gradient caused by

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root absorption. Mass flow occurs in a wide range and over a long distance, e.g., the movement of nutrients from non-rhizosphere to rhizosphere soil (Matimati et al., 2013).

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Therefore, the movement of Se from non-rhizosphere to rhizosphere soil occurred through mass flow in this experiment. Compared with the low-Se cultivar, the high-Se cultivar possessed a higher rhizosphere Se concentration, and a lower non-rhizosphere Se concentration in the 5.0 mg Se kg-1 treatment. However, there were no significant differences

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in the rhizosphere and non-rhizosphere soil Se concentrations between the two rice cultivars at the 0 and 0.5 mg kg-1 Se levels, due to the low available Se concentration within soil. It could be concluded that the high-Se rice cultivar could obtain more Se via the mass flow process, and that this process was also affected by the concentration of mobile Se within the soil. The application of 5.0 mg kg-1Se decreased total organic acid secretion by 81.6 % and 47.3 %

in the high- and low-Se cultivars, respectively. Therefore, the secretion of organic acids by the high-Se cultivar was more sensitive to the high Se concentration within the soil, than that of the low-Se cultivar. Previous studies have shown that the secretion of organic acids by roots is an important mechanism for alleviating nutrient fixation in the soil (Aziz et al., 2014; Clarholm et al., 2015). However, the relationship between organic acid secretion and soil Se availability has not been clearly demonstrated. In the present research, the secretions of oxalic acid, tartaric acid, malic acid, malonic acid and acetic acid were negatively correlated to the

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Se concentrations in the rhizosphere soil of the two rice cultivars. Overall, the secretion of organic acids decreased with the increase of Se application. Although the 0 mg kg-1Se

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treatment was most conductive to increase the secretion of organic acids in the two rice

cultivars, the secretion of organic acids did not significantly increase the proportion of watersoluble and exchangeable Se.

Selenite, which was applied in this experiment, is the major speciation present in acidic and

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neutral soils; the selenite speciation is adsorbed to soil and is generally unavailable for plant

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uptake (Li et al., 2015). Thus, the improvement of soil pH is an important way to increase the

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availability of Se in soil. The pH of rhizosphere soil was higher than that of the nonrhizosphere soil, which indicated that the availability of Se in rhizosphere soil of the two rice

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cultivars was increased by the activity of root system. Furthermore, the pH of rhizosphere soil of the high-Se cultivar was significantly higher than that of the low-Se cultivar in the 0 mg Se

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kg-1 treatment. The result showed that the high-Se cultivar had a greater ability to activate Se by increasing soil pH than the low-Se cultivar, especially in the treatment with no Se.

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However, the positive correlation between pH and organic acid secretion also revealed that organic acid secretion could not due to increase pH to enhance the availability of soil Se in

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this experiment.

Kaolinite is a dominant clay mineral in the soils of tropical and subtropical regions, and its dissolution has an influence on a variety of soil properties (Jacobs, 2017). Kaolinite-type clay mineral has the ability to fix Se, and thus its presence can decrease the bioavailability of Se

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within the soil (Yang et al., 2012; Ervanne et al., 2016). The concentration of kaolinite can be characterized by the intensity of the infrared spectrum (Zhao et al., 2017). The infrared absorption spectrum of rhizosphere soil was observed in this research, and the characteristic curve shows that it corresponds to the absorption peak of kaolinite. This result revealed that kaolinite is the main component of clay mineral within the test soil, and its content can affect the availability of Se in the soil. The infrared absorption intensities of rhizosphere soils of the two rice cultivars were increased by the application of Se, and the high-Se cultivar showed a

higher rate of increase than the low-Se cultivar. In contrast, organic acid secretions of the two rice cultivars were decreased by the application of Se and the high-Se cultivar exhibited a greater degree of reduction than the low-Se cultivar. These results revealed that there would be an internal connection between organic acid secretion and clay mineral concentration. The influence of organic acids on the kaolinite-type clay mineral was strongly supported by previous studies. The mechanism proposed by previous studies for organic acid enhancement

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of kaolinite dissolution showed that anions of organic acids could form complexes with Al on the kaolinite surface and promote its dissolution (Hu et al., 2005; Hu et al., 2007; Li et al.,

2012). The enhanced rate of kaolinite dissolution is related to the complexing ability of the

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anions of the kind of organic acid. Previous studies have shown that the ability of different organic acids to mobilize Al follow the order: oxalic acid > malonic acid > malic acid > tartaric acid > acetate acid (Chin et al., 1991; Zhang and Bloom, 1999; Wang et al., 2005;

Kong et al., 2014). This order was in agreement with the magnitude of the stability constants

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of Al-organic acid complexes. Oxalic, malonic, malic, tartaric, and acetate acids are the main

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components of organic acids in root exudates of rice. In this experiment, the effect of organic

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acids on the kaolinite-type clay mineral was also supported by the intensity of the infrared absorption spectrum. The treatment of 0 mg kg-1 Se is most beneficial to the secretion of

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organic acids in the two rice cultivars, while the reduced intensity of the infrared spectrum showed that the kaolinite-type mineral underwent a stronger dissolution than the other

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treatments. It could be concluded that the secretion of organic acids may be a mechanism of alleviating Se deficiency. Although we have not observed a significant effect of organic acid

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secretion on increase soil available Se concentration, we should face up to the fact that rice increases the secretion of organic of acids and then promotes the degradation of the clay mineral so as to reduce the fixation of Se. The secretion of organic acids in the high-Se

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cultivar was more sensitive to the change in Se concentration within the soil, than that of the low-Se cultivar, thus, the high-Se rice cultivar may have a greater ability to regulate the secretion of organic acids.

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In conclusion, the grain Se concentration of the high-Se rice cultivar was significantly higher than that of the low-Se rice cultivar, and the difference in grain Se concentration between the two rice cultivars was derived not only from the process of absorption, but also from transport. The high-Se rice cultivar could obtain more Se than the low-Se cultivar by mass flow at the high Se treatment. The high-Se cultivar had a greater ability to activate Se by increasing soil pH, compared to the low-Se cultivar. Furthermore, the secretion of organic acids could activate Se by degrading clay mineral, and the high-Se rice cultivar may have a

greater ability to regulate the secretion of organic acids. Therefore, the high-Se rice cultivar has a stronger ability to regulate the availability of Se in rhizosphere soil than the low-Se cultivar.

Acknowledgements This work was supported by the National Natural Science Funds of China (Grant No. 31501835; 31872176), the Natural Science Foundation of Guangdong Province (Grant

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No.2015A030310449) , the Science and Technology Project of Guangzhou (Grant No.

201804010341) and the Dean Fund of the Guangdong Academy of Agricultural Sciences

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(Grant No. 201435).

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Figure and table captions: Figure 1 The sketch of the root box used in the experiment. Figure 2 Effect of Se on the grain yield and plant fresh weight in the two rice cultivars. The bars indicate the standard error of the mean. Significant differences in the mean value of each treatment group are indicated by different lowercase letters based on the LSD-test (p<0.05,

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n=4). Figure 3 Effect of Se on the root, shoot and grain Se concentrations in the two rice cultivars.

The bars indicate the standard error of the mean. Significant differences in the mean value of

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each treatment group are indicated by different lowercase letters based on the LSD-test (p<0.05, n=4).

Figure 4 Effect of Se on the organic acid secretions in the two rice cultivars. The bars indicate

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the standard error of the mean. Significant differences in the mean value of each treatment

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group are indicated by different lowercase letters based on the LSD-test (p<0.05, n=4). Figure 5 Effect of Se on the pH of rhizosphere and non-rhizosphere soils of the two rice

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cultivars. The bars indicate the standard error of the mean. Significant differences in the mean

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value of each treatment group are indicated by different lowercase letters based on the LSDtest (p<0.05, n=4).

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Figure 6 Effect of Se on the total Se concentration of the rhizosphere and non-rhizosphere soils of the two rice cultivars. The bars indicate the standard error of the mean. Significant

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differences in the mean value of each treatment group are indicated by different lowercase letters based on the LSD-test (p<0.05, n=4).

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Figure 7 Effect of Se on the water-soluble, exchangeable, Fe/Mn oxide-bound, organic matter/sulphide-bound and residual Se concentrations of the rhizosphere and non-rhizosphere soils of the two rice cultivars. The bars indicate the standard error of the mean. Significant differences in the mean value of each treatment group are indicated by different lowercase

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letters based on the LSD-test (p<0.05, n=4). Figure 8 Effect of Se on the proportions of water-soluble, exchangeable, Fe/Mn oxide-bound, organic matter/sulphide-bound and residual Se concentrations of the rhizosphere and nonrhizosphere soils of the two rice cultivars. The bars indicate the standard error of the mean. Significant differences in the mean value of each treatment group are indicated by different lowercase letters based on the LSD-test (p<0.05, n=4).

Figure 9 Effect of Se on the infrared absorption spectrum of rhizosphere soils of the two rice cultivars. .

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Figure 1

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Figure 8 Exchangeable Water soluble Organic matter and sulfide bound 15.3%

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Figure 9

1031.95 912.89 796.52 694.51 535.93 469.98

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400 4000 3600 3200 2800 2400 2000 1600 1200 800

-1

400 4000 0 3600 3200 2800 2400 2000 1600 1200 800

Wave number (cm-1)

-1

Wave number (cm )

A

CC E

PT

ED

M

A

N

U

SC R

Wave number (cm )

400

0

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Table 1 Pearson correlation coefficients for relationship between rhizosphere soil and plant items Items Plant Se Organic acids Tartaric Malic Malonic Acetic Grain Leaf Root Oxalic acid acid acid acid acid H-Se Se levels 1.00** 0.99** 0.99** -0.89** -0.75** -0.87** -0.94** -0.95** TS 0.99** 0.98** 0.99** -0.89** -0.75** -0.87** -0.94** -0.94** WS 1.00** 0.99** 0.98** -0.90** -0.71* -0.87** -0.95** -0.95** EX 0.94** 0.97** 0.91** -0.81** -0.68* -0.76** -0.84** -0.90** FM 0.96** 0.98** 0.93** -0.83** -0.69* -0.79** -0.87** -0.90** OM 0.84** 0.87** 0.80** -0.66* -0.53 -0.58 -0.71* -0.77** RE 0.95** 0.92** 0.92** -0.85** -0.72* -0.86** -0.91** -0.90** pH -0.66* -0.67* -0.64* 0.49 0.85** 0.25 0.51 0.67* L-Se Se levels 1.00** 0.99** 0.99** -0.69* -0.77** -0.67* -0.77** -0.72* TS 0.97** 0.95** 0.97** -0.68* -0.78** -0.69* -0.79** -0.73* WS 0.98** 0.99** 0.97** -0.64* -0.72* -0.62* -0.74** -0.68* EX 0.94** 0.94** 0.93** -0.71* -0.77** -0.69* -0.78** -0.68* FM 0.97** 0.97** 0.96** -0.67* -0.76** -0.67* -0.77** -0.71* OM 0.96** 0.95** 0.95** -0.68* -0.75** -0.67* -0.77** -0.68* RE 0.95** 0.92** 0.92** -0.64* -0.75** -0.66* -0.75** -0.73* pH -0.84** -0.82** 0.78** 0.89** 0.76** 0.74** 0.49 0.83** Note: TS-Total Se in rhizosphere soil, WS-Water soluble Se, EX-Exchangeable Se, FM-Fe/Mn oxide-bound Se, OM-Organic matter/sulfide-bound Se, RE-Residual Se. * means the significant level at P<0.05 and ** mean the significant level at P<0.01.