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Effects of Fe-oxidizing bacteria (FeOB) on iron plaque formation, As concentrations and speciation in rice (Oryza sativa L.)
Ecotoxicology and Environmental Safety 190 (2020) 110136
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Effects of Fe-oxidizing bacteria (FeOB) on iron plaque formation, As concentrations and speciation in rice (Oryza sativa L.)
T
Anwen Xiaoa, Wai Chin Lib,∗,1, Zhihong Yea,∗∗ a b
School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China Department of Science and Environmental Studies, The Education University of Hong Kong, Hong Kong, SAR, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fe-oxidizing bacteria Rice Arsenic Speciation
Large areas of the paddy fields in South China are contaminated with arsenic (As), which causes serious problems, including high As concentrations in brown rice. Three As-resistant iron-oxidizing bacteria (FeOB) namely, Bacillus sp. T2, Pseudomonas sp. Yangling I4 and Bacillus sp. TF1-3, were isolated and applied to rice grown in different As-contaminated environments to study the effects of FeOB on the As accumulation in rice and clarify the possible mechanisms involved. The results showed that FeOB inoculation significantly decreased the inorganic As concentrations in brown rice grown in pots and paddy fields by 3.7–13.3% and 4.6–12.1%, respectively. FeOB inoculation enhanced the formation of Fe plaque, which sequestered more As on the root surface. Moreover, a significantly lower level of As(III) influx was observed in the rice cultivated with FeOB than in the control. FeOB inoculation also decreased the As concentrations in pore water and the Fe(II)/Fe(III) ratio in rhizosphere soil. The present results suggest that FeOB inoculation decreased the inorganic As concentrations in brown rice by affecting the formation of Fe plaque, As(III) uptake kinetics and rhizosphere soil properties. Based on our results, FeOB inoculation could be considered a useful method to decrease inorganic As concentrations in brown rice grown in As-contaminated paddy fields.
1. Introduction
accounting for 70%–88% of the total As in rice grains (Li et al., 2015; Lin et al., 2015). The predominant As species in agricultural soils are iAs, and there is a higher As(III) bioavailability than other As species in flooded paddy fields, which results in more iAs in rice grains (Du et al., 2019). In other words, compared to the total As, we must pay close attention to the iAs concentration in brown rice. Iron (Fe) plaque is an important barrier for roots of wetland plants and prevent damage inflicted by reductive substances on plants after flooding. Moreover, Fe plaques are also a potential barrier for plants exposed to As (Tripathi et al., 2014). In the present study, much lower As concentrations were measured in Typha root tissues than in the Fe plaque on the root surface. Arsenic was spatially located with Fe, indicating that As was absorbed by Fe in Fe plaque (Blute et al., 2004; Mei et al., 2012). In a hydroponic experiment, Fe plaque on rice root surface functioned as a barrier to As and reduced As accumulation in the root and shoot (Wu et al., 2012). In paddy fields, the formation of Fe plaque on the root surface limits As uptake by rice, and most of the As released from soils was distributed in Fe plaque compared to rice tissues (Syu et al., 2013).
Large areas of paddy fields have been contaminated with arsenic (As) because of mining and other human activities in Asia (Williams et al., 2009). Arsenic exerts very toxic effects on plant growth when its concentrations exceed a threshold (Ünyayar et al., 2006). High levels of As in paddy soils result in higher As concentrations in brown rice (Mei et al., 2009; Zhuang et al., 2014). As the most toxic speciation of As, inorganic As (iAs) [As(III), As(V)] is a non-threshold carcinogen, while organic As is less toxic (Batista et al., 2011). Inorganic As exposure can cause skin, lung, and bladder cancers and may result in non-cancerous respiratory, cardiovascular, metabolism and nervous system diseases (Sanchez et al., 2016). Compared to total As concentration, iAs concentration is a better index to estimate the health risk of As in rice (Kumarathilaka et al., 2019; Lin et al., 2015). The World Health Organization standard of iAs in polished rice grains for adults is 200 μg/kg (World Health Organization, 2014). Moreover, investigations in China have demonstrated that most of the As in the rice grains grown in As-contaminated paddy fields is iAs,
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (W.C. Li), [email protected] (Z. Ye). 1 Dr. LI WC will handle correspondence at all stages of refereeing and publication, also post-publication. ∗∗
https://doi.org/10.1016/j.ecoenv.2019.110136 Received 14 August 2019; Received in revised form 24 December 2019; Accepted 24 December 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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citrate, 0.5 g of MgSO4·7H2O, 0.5 g of (NH4)2SO4, 0.5 g of K2HPO4, 0.5 g of NaNO3, 0.2 g of CaCl2, and 1 L of distilled water) (Ghosh et al., 2014; Zhou et al., 2011). After incubation, colonies with varying morphologies were selected from the plates and streaked on the same medium at least five times to purify the clone. The strains were then maintained as glycerol stocks stored at −80 °C (Singh et al., 2016a).
Iron plaque affect the rate of As absorption by the roots. Chen et al. (2005) reported that Fe plaque on the rice root surface increased the rate of As(III) absorption but decrease the rate of As(V) absorption. In another hydroponic experiment, Deng et al. (2010) also found that Fe plaque increased the As(III) absorption rate of rice roots, decreased As (V) absorption rate at low As(V) concentration (< 2 mg/L), but increased As(V) absorption rate at high As(V) concentration (> 6 mg/L). Because some management techniques could induce Fe plaque formation, As accumulation in rice may be reduced. For example, when the amount of added sulfur increases, the As concentration in the rice shoots decreases, which is attributed to the increased formation of Fe plaque on the root surface and in rhizosphere soil (Hu et al., 2007). Previous studies have reported that Fe-oxidizing bacteria (FeOB) promotes the formation of Fe plaque on the roots of wetland plants (Neubauer et al., 2007; Schmidt and Eickhorst, 2013). In a wetland plant (e.g., Juncus effusus) rhizosphere, high densities of lithotrophic FeOB in a favourable environment [such as high Fe(II) availability] suggests that FeOB actively contribute to the formation of Fe plaque on plant roots (Neubauer et al., 2007). The abundance of FeOB in the rhizosphere area correlates with Fe content on the root surface and suggests that FeOB plays important roles in the formation of Fe plaque (Begg et al., 1994; Emerson et al., 1999; Schmidt and Eickhorst, 2013). Moreover, artificial treatment also shows that FeOB inoculation increases the formation of Fe plaque on the surfaces of reed roots (Guo and Cutright, 2015). FeOB have a close relationship with As and Fe levels in soil. In axenic hydroponic microcosms, the presence of FeOB in the J. effusus rhizosphere increases the short-term rates of Fe(II) oxidation by 1.3–1.7 times compared to a control (Neubauer et al., 2007). As shown in the study by Neubauer et al. (2002), biotic oxidation of FeOB competes with abiotic Fe(II) oxidation and accounts for up to 60% of the total Fe (II) oxidation in laboratory cultures. Moreover, the growth of FeOB stimulates nitrate-dependent Fe(II) oxidation, leading to As co-precipitation or adsorption to Fe(III) minerals in the soil, which sequesters the mobile As in the soil and results in a reduced As uptake by rice (Chen et al., 2008). Previous studies show that Fe plaque on rice root surface reduce As accumulation, therefore, FeOB, which promotes the formation of Fe plaque implies that FeOB inoculation may affect As accumulation in rice grown in As-contaminated soils. However, studies on the influence of FeOB on Fe plaque formation and As accumulation in rice grown in As-contaminated environments are rare. Only one study showed that FeOB inoculation increased Fe plaque formation and decreased As accumulation in rice tissues in a pot experiment (Dong et al., 2016). Unfortunately, the mechanisms underlying these phenomena remain unclear, and the effect of FeOB on rice grown in paddy fields is also unknown. A series of experiments (including hydroponic, pot and field experiments) were conducted to answer these questions. The aims of this paper are to investigate the effects of FeOB on As (including iAs) accumulation in rice, elucidate the possible mechanisms involved and explore whether FeOB represents a useful modification to As-contaminated paddy soil.
2.1.1. Arsenic resistance ability All strains were cultivated onto pH 7.0 FeOB media containing 1000 mg/L As(V), placed in a 28 °C incubator shaker for 2 days, and the growth of the strains was determined by measuring the absorbance at 600 nm using the method reported by Hohmann et al. (2010). The FeOB media that were not inoculated with a strain were used as a reference. 2.1.2. Fe oxidation ability All strains were cultivated onto pH 7.0 FeOB media and then placed in a 28 °C incubator shaker for 7 days. The FeOB media from the upper layer was collected and filtered through a 0.45 μm membrane filter (MICROPORE, Minigene Syringe Filter, Genetix Biotech Asia Pvt. Ltd) to remove the particulate material. Phenanthroline photometry was used to determine Fe(II) concentrations (Zhou et al., 2011). Five millilitres of an acetic acid-ammonium acetate buffer solution and a 0.5% 1,10-phenanthroline solution were added to a 50 mL sample, and after 10 min, the absorbance of the mixed solution was determined at 510 nm. Distilled water was used for a reference. 2.1.3. As fixation ability All strains were cultivated onto FeOB media that contained 1 mg/L As(V) and then placed in a 28 °C incubator shaker for 7 days. The FeOB media were filtered, and the As concentration was determined using atomic fluorescence spectrometry (AFS, Beijing Titan Instrument Co. Ltd.) (Hohmann et al., 2010). 2.1.4. Indole acetic acid (IAA) This experiment was performed using the methods described by Mahmood et al. (2017). Bacterial cultures were grown in a Pikovskaya medium with tricalcium phosphate under standard conditions for 120 h to determine the solubilization of phosphates (Iyer et al., 2017). The supernatants from the cultures were centrifuged at 12,857×g for 10 min. The concentration of soluble phosphate in the supernatant was estimated using the phosphomolybdate blue method (Islam et al., 2014). 2.1.5. Identification by 16S rRNA gene sequencing The above strains that were identified with a relatively higher tolerance were selected for genetic identification. Genomic DNA was extracted, and the 16S rRNA gene was amplified by PCR using the procedures described by Chun and Goodfellow (1995). The universal primers for the bacterial 16S rRNA gene (the forward primer P1 5′-CGG GAT CCA GAG TTT GAT CCT GGC TCA GAA CGA ACG CT- 3′ and the reverse primer P6 5′-CGG GAT CCT ACG GCT ACC TTG TTA CGA CTT CAC CCC-3′) were used to amplify the target gene (Ben-Dov et al., 2006). The PCR product was purified and directly sequenced with an automated DNA Sequencing System (ABI 3730XL). Almost complete 16S rRNA gene sequences were obtained and matched, using the BLASTn program (http://blast.ncbi.nlm.nih.gov/), to nucleotide sequences in the GenBank, European Molecular Biology Laboratory (EMBL) and the DNA Data Bank of Japan (DDBJ) databases.
2. Materials and methods 2.1. FeOB isolation, identification and characterization The rice rhizosphere soil samples were collected from an As-contaminated paddy field near the Baoshan mine (112°44′18.64″E, 25°43′4.38″N), Chenzhou City, Hunan Province, China, which has been described by Liao et al. (2005). The soil samples (1 g) were suspended in 50 mL of a modified Wolfe mineral media at a pH of 6.0 and incubated at 37 °C for 30 min (Ghosh et al., 2014). The supernatant was filtered through a 0.45 μm membrane filter (MICROPORE, Minigene Syringe Filter, Genetix Biotech Asia Pvt. Ltd). For isolating FeOB, the filtrate was plated onto pH 7.0 FeOB media (10 g of ferric ammonium
2.2. Inoculum and seedling preparation The selected rhizobacterial isolates were grown in FeOB medium at 28 °C for 1 day. Bacterial cells were harvested by centrifugation and resuspended in distilled water prior to inoculation. The rice cultivar Jianyou G2 (which is widely cultivated in Guangdong Province) was chosen, and the seeds were purchased from 2
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Golden Rice Seeds Co. Ltd. of Guangdong. Seeds were surface sterilized with a 30% H2O2 solution for 15 min and washed with deionized water. For hydroponic and pot experiments, the seeds were germinated in sterilized petri dishes containing moist filter paper in a controlled chamber (28 °C and 70% relative humidity) (Wu et al., 2011). 2.3. Analysis of the uptake kinetics The study of As uptake kinetics in roots was performed using the method described by Li et al. (2011). Three-week-old rice seedlings were incubated with (i) a bacterial suspension in sterile distilled water (109 cfu/mL) or (ii) sterile distilled water for 1 h as control (Dong et al., 2016; Singh et al., 2016a, 2016b). After different treatments, the seedlings were excised, and then the roots were incubated for 40 min with test solutions that contained arsenite (NaAsO2) or arsenate (Na2HAsO4·7H2O). All test solutions contained 5.0 mM 2-(N-morpholino)-ethanesulfonic acid (MES) and 0.5 mM Ca(NO3)2, with the pH adjusted to 5 using KOH. Next to the roots were rinsed for 15 min with an ice-cold phosphate solution that contained 1 mM K2HPO4, 5 mM MES and 0.5 mM Ca(NO3)2 to remove the absorbed As species from the root free space. Finally, the roots were washed and oven-dried at 60 °C and then the As concentrations in the root tissues were analysed. 2.4. Hydroponic experiment Uniform rice seedlings were transplanted to plastic vessels and incubated with a ¼ dilution of Hoagland's nutrient solution (Hoagland μmol NaHAsO•7HO for 30 d and Arnon, 1938) and 40 . The nutrient solutions 2 4 2 were replaced once every 3 days during the growth period. All vessels were arranged randomly in a controlled greenhouse with natural light supplemented by sodium light and a day/night temperature of 25/ 20 °C, a day/night photoperiod of 12/12 h and a relative humidity of 70%. Seedlings of each cultivar were incubated with (i) a bacterial suspension in sterile distilled water (109 cfu/mL) or (ii) sterile distilled water for 1 h as control (Dong et al., 2016; Singh et al., 2016a, 2016b). Four replicates were prepared for each treatment. After 36 days, the plants were carefully washed with deionized water and separated into shoots and roots. All samples were freeze-dried and milled to a powder for concentration analyses of the total As and As species [arsenite (As (III)), arsenate (As(V)), dimethylarsinate (DMA), and monomethylarsonate (MMA)].
Fig. 1. Grain yield of rice grown in pots with As-contaminated soils collected from Dabaoshan (a) and paddy fields at Dabaoshan (b) with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments within the same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
2.5. Pot experiment
2016b). Pore water was collected at the rice heading stage using rhizon soil pore water samplers (Rhizosphere Research Products, The Netherlands), as previously reported by Kidd et al. (2007). Rice plants were harvested at the mature stage and separated into roots, straw and brown rice. All plant samples (except roots) were washed, freeze-dried at −50 °C, milled to a fine powder, and stored at 4 °C until further analysis. Roots were divided into two subsamples: one for the enzyme analysis and another for the total As analysis. The total concentrations of As in rice tissues and pore water were analysed using AFS after microwave digestion. The concentrations of As species in the rice were determined by weighing 0.4 g of rice flour into a 10 mL polypropylene tube and extracting the sample with 8 mL of 1% HNO3 at 90 °C for 2.5 h (Lin et al., 2015). The extract was then centrifuged at 8228×g for 10 min at 4 °C, and the supernatant was filtered
For the pot experiment, the soil was collected from the surface (0–20 cm) of two As-contaminated paddy fields in Dabaoshan (DBS, 113°49′5.6″E, 24°28′44.1″N), Shaoguan City, Guangdong Province, and named DA and DB, respectively. The basic soil properties are shown in Table 1. A cylindrical nylon rhizobag (30 μm nylon mesh, 12 cm in diameter and 15 cm in height) was designed to separate the rhizosphere soil from the bulk soil (Wang et al., 2014). Uniform rice seedlings were carefully transplanted into each rhizobag. Four replicates were prepared for each treatment (Wang et al., 2014). FeOB (as a bacterial suspension in sterile distilled water, 109 cfu/mL) and sterile distilled water (control) were applied to the rhizobag three times (at the transplanting, tillering and heading stages, respectively) (Dong et al., 2016; Singh et al., 2016a, Table 1 Basic properties of soils collected from the two paddy fields (Mean ± SE, n = 6).
DA DB
As (mg/kg)
Mn (mg/kg)
Zn (mg/kg)
Cu(mg/kg)
Fe (g/kg)
K (g/kg)
P (g/kg)
pH
138 ± 8.7 41 ± 1.9
153 ± 8.2 178 ± 5.0
320 ± 19 292 ± 17
370 ± 20 408 ± 26
38 ± 2.2 29 ± 0.4
28 ± 1.1 26 ± 2.0
11 ± 0.83 12 ± 0.51
5.22 ± 0.21 5.47 ± 0.18
Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. 3
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Fig. 2. Arsenic concentration in brown rice grown in pots with DA (a) and DB (b) soil and in DA (c) and DB (d) paddy fields and DMA concentration in brown rice grown in pots (e) and fields (f) with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments within the same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3. 4
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Fig. 3. The concentration of As in shoots (a) and roots (b) in pot experiments with different FeOB strains inoculation and As concentration in shoots (c) and roots (d) in field experiments with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments within the same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
through a 0.22 μm polyethersulfone membrane (Tianjin Branch Billion Lung Experimental Equipment Co. Ltd. Tianjin, China). The samples were stored at 4 °C and analysed within a few hours after extraction to minimize the transformation of As species. Arsenic speciation in the rice extracts was performed using atomic fluorescence spectrometry-highperformance liquid chromatography (AFS-HPLC, Beijing Titan Instrument Co. Ltd.) (Chi et al., 2018). The dithionite-citrate-bicarbonate (DCB) method (Otte et al., 1991; Taylor and Crowder, 1983) was used to extract Fe plaque on the fresh root surfaces of rice plants. Fresh roots were extracted for 3 h in a solution of 30 mL of 0.03 M Na3C6H5O7·2H2O, 0.125 M NaHCO3 and 0.6 g of Na2S2O4 at room temperature (25 °C). After extraction, the roots were washed 3 times with 15 mL of deionized water, and the wash was collected. The volumes of the resulting extracts were brought to 50 mL with deionized water, and then the concentrations of Fe and As were analysed. The soil within the rhizobags was regarded as the rhizosphere soil (Wang et al., 2014). The rhizosphere soil samples were freeze-dried at −50 °C, crushed, sieved through a 150 mesh, and collected to measure the levels of extractable As, the pH and the soluble P. The pH was measured with a glass electrode (soil: water = 1:2.5) (Lu, 1999). The As concentrations from the soil extract were measured using AFS after a 2 h extraction in leach liquor (0.01 M CaCl2, solution: soil = 5:1) (Li et al., 2014). The Fe(II)/Fe(III) ratio was calculated using the method reported by Begg et al. (1994). The rice rhizosphere soil was collected and approx. 1 g of soil was placed in a tube containing 3 mL of 5 mM CaCl2 under an anaerobic environment. After the addition 10 mL of 1 M NH4OAc (pH
2.8), each tube was sealed and shaken for 30 min. The extracts were centrifuged at 1575×g for 10 min. The supernatant was filtered through a 0.45 μm membrane filter and used for the Fe(II) assay. Ten millilitres of 2 M H2SO4 was added to the residue, and then the tube was shaken for 30 min. The extracts were centrifuged at 1575×g for 10 min and the filtered supernatant was used for the Fe(III) assay. 2.6. Field experiments Field experiments were conducted at two paddy fields in Dabaoshan, as mentioned above. The rice cultivar Jianyou G2 was chosen. Each treatment included four replicates with a 3 m × 3 m plot (Wang et al., 2014). All plots were randomly located in each paddy field. Two rice seedlings were carefully transplanted into each rhizobag. FeOB inoculation and control conditions were similar to the pot experiment. Field management was performed according to the strategies employed locally, except for the addition of FeOB. Rice samples were harvested at the mature stage, and the sample treatment methods were similar to the pot experiment. 2.7. Quality control and statistical analyses Blanks, soil standard material (GBW-07401), plant standard material (GBW-07435) and brown rice standard material (GBW-10013) (China Standard Materials Research Centre, Beijing, P.R. China) were used for quality control. Recoveries from the reference materials ranged from 85.1% to 92.0% for the total As analysis and 87.0%–91.2% for the iAs. 5
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Fig. 4. The concentration of Fe (a) and As (b) in Fe plaque on root surface in pot experiments with different FeOB strains inoculation and the concentration of Fe (c) and As (d) in Fe plaque on root surface in field experiments with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments in same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
In the hydroponic experiment, the As concentration in the shoots of rice with F1 inoculation was reduced by 42.6%–63.5% (Fig. S6a). The total As concentration in the roots was significantly decreased by FeOB (P < 0.05), and the As(III) and As(V) concentrations were also obviously decreased by 10.8%–36.4%, after FeOB inoculation (Fig. S6b). Interestingly, the roots of rice grown in hydroponic culture contained only iAs, while the shoots contained As(III), As(V) and DMA (Fig. S6). Compared with the control, the total As concentration in brown rice with F3 and F10 inoculation was significantly reduced by 15.7% and 18.9%, respectively, in pots with DA soil (Fig. S7a). As for As species, the FeOB (except F3) significantly decreased the iAs concentration in brown rice grown in pots by 3.7%–13.3% (P < 0.05), which was attributed to the decrease in As(III) concentration by 4.0%–16.3% (Fig. 2a and b). Regarding the organic As concentration in brown rice, FeOB inoculation significantly decreased the DMA concentration in brown rice grown in pots with DA soil by 18.9%–36.0% (P < 0.05) (Fig. 2e). In DB paddy field experiment, total As in brown rice with F1 and F10 inoculation also significantly decreased by 11.4% and 17.5%, respectively (Fig. S7b). The FeOB did not affected As concentration in brown rice grown at DA paddy field, however, the FeOB could significantly decreased the iAs concentration in brown rice by 8.4%–12.1% under DB field conditions (P < 0.05) (Fig. 2c and d). In field experiments, F1 and F10 inoculation significantly decreased the DMA concentration in brown rice grown at DB by 21.4% and 36.1%, respectively (P < 0.05) (Fig. 2f). In pot experiments, FeOB inoculation significantly increased As concentrations in rice shoots and roots by 13.3%–137.4% and 11.9%–57.8%, respectively (P < 0.05) (Fig. 3a and b). However, the
Origin 8.0 and Excel 2007 software were used to create the artwork. Data were analysed using the SPSS 19.0 statistical software package and summarized as the means ± standard error (SE). Treatment means were compared using the Duncan multiple comparisons test at the 5% level of significance. 3. Results 3.1. The isolated FeOB Twelve FeOB were isolated from the rhizosphere soil of rice grown in an As-contaminated paddy field. Based on their Fe oxidation ability, As resistance, As fixation ability, phosphate solubilizing and IAA production capacity, three FeOB (F1, F3, and F10) were selected, and some of their properties are shown in Figs. S1–S5. Compared to the other strains, the three selected FeOB all exhibited a higher As resistance, Fe oxidation ability and As fixation ability (Figs. S1–S3). The results from physiological and biochemical analyses and 16S rDNA sequencing showed that F1 was Bacillus sp. T2, F3 was Pseudomonas sp. Yangling I4 and F10 was Bacillus sp. TF1-3. 3.2. The effect of FeOB on the grain yield and As accumulation in rice The grain yield with F1 and F10 inoculation increased by 40.1% and 25.6%, respectively, in pots with the DB soil (Fig. 1a). However, the grain yield of rice grown in pots with DA soil and in two paddy fields did not differ significantly between the plants with and without FeOB inoculation (Fig. 1b). 6
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particularly in roots cultured with F3. 3.4. Soil properties In the pot experiments, compared to the control, the Fe(II)/Fe(III) ratio in rhizosphere soil of rice plant inoculated with FeOB decreased, particularly for the plant inoculated with F1 and F10, which significantly decreased the Fe(II)/Fe(III) ratio by 7.7%–44.2% (P < 0.05), except for F1 in DA (Fig. 6a). The FeOB did not substantially alter the rhizosphere soil pH or the available As concentration in soils of the pot trials (Fig. 6b and c). However, following F1 and F10 inoculation, As concentrations in soil pore water were significantly reduced by 26.7%–55.0% (P < 0.05), except for the DA inoculated with F10 (Fig. 6d). 4. Discussion According to the 16S rRNA gene sequencing data, the three isolated FeOB belong to Pseudomonas (F3) and Bacillus (F1 and F10), respectively. These results were consistent with a recent study indicating that some FeOB belong to these two genera (Ghosh et al., 2014). 4.1. As concentration in rice plants Compared to the total As, the iAs concentration in brown rice requires more attention because its toxicity is higher than that of other As species, and iAs is a better index than total As (Batista et al., 2011; Kumarathilaka et al., 2019; Lin et al., 2015). In the present study, iAs concentration in brown rice decreased following FeOB inoculation in all hydroponic, pot and field experiments, particularly after the inoculation of F1 and F10 (Fig. 2 and S6), indicating that FeOB inoculation could effectively reduce the risk of food safety hazards from rice grown in As-contaminated paddy fields. In a previous study, Dong et al. (2016) reported that the total As concentration in brown rice decreased following FeOB inoculation in a pot experiment. The explanations for this decrease are related to the changes in soil properties and the rice plants following FeOB inoculation. On the one hand, As concentrations in soil pore water were significantly decreased following the F1 or F10 treatment, which may be due to the decrease in the Fe(II)/Fe(III) ratio in the rhizosphere soil inoculated with F1 and F10 (Fig. 6). Consistent with the present results, some previous studies also found that FeOB increased Fe(II) oxidation rates in soil and that the growth of FeOB sequestered more mobile As due to increased Fe(III) formation (Chen et al., 2008; Neubauer et al., 2007). All three selected strains decreased mobile As and Fe concentrations in FeOB media (Figs. S2 and S3). On the other hand, compared to the control, significantly lower As (III) influx was observed following FeOB inoculation (P < 0.05) (Fig. 5a), which was consistent with the decrease in As(III) concentrations in brown rice (Fig. 2). In addition, in DA, the concentrations of Fe and As in Fe plaque increased with FeOB inoculation (Fig. 4), indicating that FeOB inoculation promoted Fe plaque formation and then fixed more As in the Fe plaque, which contributed to the decrease of iAs concentration in the brown rice. A previous study reported that FeOB promotes Fe plaque formation on the roots of wetland plants (Neubauer et al., 2007), which was consistent with the results of the present study (Fig. 4). The increased formation of Fe plaque on the root surface may account for the decrease in As(III) uptake by rice roots cultured with FeOB. Iron plaque affect As uptake by plant roots, and some of the As is fixed in the Fe plaque, reducing the As accumulation in plant tissues (Blute et al., 2004; Deng et al., 2010; Syu et al., 2013). Moreover, the decreased rate of As transfer in rice tissues may contribute to the decrease of As concentrations in brown rice, particularly in the DA soil (Table S1). The effects of FeOB inoculation on As concentration in shoot and root tissues were different between the rice grown in the pots and in the
Fig. 5. Concentration-dependent kinetics for As(III) (a) and As(V) (b) uptake by rice roots with different FeOB strain suspension. (Mean ± SE, n = 4). Note: CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
As concentrations in the shoots and roots of rice grown in paddy fields inoculated with FeOB (F1 and F10) did not significantly differ from the control (Fig. 3c and d). 3.3. The Fe plaque and As uptake kinetics of rice roots In pot experiments, FeOB inoculation increased Fe concentrations in Fe plaque on root surfaces and a significant increase was noticed in DA (11%–28.1%) (P < 0.05) (Fig. 4a). Moreover, the As concentrations in Fe plaque also increased following FeOB inoculation by 14.6%–42.5%, except for the inoculation of F1 in DB (Fig. 4b). In both DA and DB paddy field experiments, following FeOB (F1 and F10) inoculation, concentrations of Fe and As in Fe plaque were significantly increased by 42.5–50.8% and by 46.1–66.6%, respectively (P < 0.05) (Fig. 4c and d). A significantly lower As(III) influx (decreased by 37.7%–74.6%) was observed in rice roots incubated with FeOB than in roots without FeOB (P < 0.05) (Fig. 5a). The influx of As(V), for which the external concentrations ranged from 0.02 to 0.12 mM, in roots cultured with FeOB was significantly higher than in the control (Fig. 5b) (P < 0.05), 7
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Fig. 6. Fe(II)/Fe(III) (a), pH(b), extracted As concentration(c) and As concentration in pore water(d) in rhizosphere soil of rice with different FeOB strains inoculation in pot experiment. (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A soil and Dabaoshan-B soil, respectively. Data with different letters mean significant differences between the treatments in same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
paddy fields. FeOB inoculation increased As concentrations in the shoots and roots of rice in the pot trials; however, the results were different in the paddy field experiments (Fig. 3). A potential explanation was that rice grown in pots absorbed more As than rice grown in fields (Figs. 2 and 3), which altered the effect of the FeOB.
to the plant growth-promoting traits of FeOB (Fig. 5b, S4 and S5). Interestingly, although FeOB increased As(V) influx in the rice roots, FeOB did not increase As(V) concentrations in the brown rice. A potential explanation for this finding was the presence of Fe plaque on the root surface and the As distribution in rice tissues. The increased As(V) influx may be mainly sequestered in Fe plaque on the root surface and in the rice roots and shoots (Figs. 3 and 4).
4.2. Rice grain yields Compared with the control, F1 and F10 inoculation significantly increased the grain yield of the rice grown in pots with DB soil (Fig. 1). The results suggested that some FeOB (e.g., F1 and F10) have some plant growth-promoting traits (e.g., phosphate solubilization and the production of IAA-like molecules) (Figs. S4 and S5). Thus, these bacteria may be a combination of FeOB and plant growth-promoting rhizobacteria, which decrease the iAs concentration in brown rice and increase the grain yield of rice grown in As-contaminated paddy fields. However, the grain yields were not significantly different between the treatments with and without FeOB inoculation in the pots with DA soil and in the paddy fields. These results suggested that the effects of FeOB on grain yields may also be regulated by other environmental factors (e.g., soil types).
4.4. The importance of environmental factors The present results showed that the effect of FeOB inoculation on As concentrations in rice tissues differed in different environments. Compared to DA, the effect of FeOB inoculation on plants grown in DB soil was more obvious, which may be attributed to the differences in soil properties, particularly the total As concentration in soil (Fig. 6 and Table 1), as the total As concentration in DB soil was significantly lower than in DA soil (P < 0.05). Based on the results from the hydroponic, pot and field experiments (Fig. 2 and S6), the difference of As concentration in rice between control and rice inoculated with FeOB was reduced. The number of influencing factors increased when the experimental environment changed from the hydroponic systems and pots to the paddy fields, making the variables more difficult to control. Moreover, during strain inoculation, inoculation in a field would dilute the influence of FeOB on rice and soil because of the abundant microbial communities in the soil compared to the pot and hydroponic experiments, which would result in a lower effect of the FeOB on the rice grown in paddy fields. Notably, researchers must be conscious of the different behaviours of FeOB in different environments and conduct
4.3. The relationship between the As(V) influx and FeOB traits Wang et al. (2011) reported that IAA stimulated the elongation of cells or increased cell division in plant roots, which resulted in increased As absorption in the roots. Therefore, compared to the control, the higher root As(V) influx in rice cultivated with FeOB may be related 8
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preliminary experiments before applying FeOB in As-contaminated paddy fields to reduce As risks.
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Mei, X.Q., Wong, M.H., Yang, Y., Dong, H.Y., Qiu, R.L., Ye, Z.H., 2012. The effects of radial oxygen loss on arsenic tolerance and uptake in rice and on its rhizosphere. Environ. Pollut. 165, 109–117. Neubauer, S.C., Emerson, D., Megonigal, J.P., 2002. Life at the energetic edge: kinetics of circumneutral iron oxidation by lithotrophic iron-oxidizing bacteria isolated from the wetland-plant rhizosphere. Appl. Environ. Microbiol. 68, 3988–3995. Neubauer, S.C., Toledo-Durán, G.E., Emerson, D., Megonigal, J.P., 2007. Returning to their roots: iron-oxidizing bacteria enhance short-term plaque formation in the wetland-plant rhizosphere. Geomicrobiol. J. 24, 65–73. Otte, M.L., Dekkers, M.J., Rozema, J., Broekman, R.A., 1991. Uptake of arsenic by Aster tripolium in relation to rhizosphere oxidation. Can. J. Bot. 69, 2670–2677. Sanchez, T.R., Perzanowski, M., Graziano, J.H., 2016. Inorganic arsenic and respiratory health, from early life exposure to sex-specific effects: a systematic review. Environ. Res. 147, 537–555. Schmidt, H., Eickhorst, T., 2013. Spatio-temporal variability of microbial abundance and community structure in the puddled layer of a paddy soil cultivated with wetland rice (Oryza sativa L.). Appl. Soil Ecol. 72, 93–102. Singh, N., Marwa, N., Mishra, S.K., Mishra, J., Verma, P.C., Rathaur, S., Singh, N., 2016a. Brevundimonas diminuta mediated alleviation of arsenic toxicity and plant growth promotion in Oryza sativa L. Ecotoxicol. Environ. Saf. 125, 25–34. Singh, N., Srivastava, S., Rathaur, S., Singh, N., 2016b. Assessing the bioremediation potential of arsenic tolerant bacterial strains in rice rhizosphere interface. J. Environ. Sci. 48, 112–119. Syu, C.H., Jiang, P.Y., Huang, H.H., Chen, W.T., Lin, T.H., Lee, D.Y., 2013. Arsenic sequestration in iron plaque and its effect on as uptake by rice plants grown in paddy soils with high contents of As, iron oxides, and organic matter. Soil Sci. Plant Nutr. 59, 463–471. Taylor, G.J., Crowder, A.A., 1983. Use of the DCB technique for extraction of hydrous iron oxides from roots of wetland plants. Am. J. Bot. 70, 1254–1257. Tripathi, R.D., Tripathi, P., Dwivedi, S., Kumar, A., Mishra, A., Chauhan, P.S., Norton, G.J., Nautiyal, C.S., 2014. Roles for root iron plaque in sequestration and uptake of
5. Conclusions In the present study, FeOB decreased As accumulation in brown rice in both pot and paddy field trials without an adverse effect on the grain yield, indicating that the application of a suitable FeOB is possible to mitigate the problem of As-contaminated brown rice after carefully selecting the appropriate strains and environment. Because the effects of FeOB were influenced by both the strains and environmental factors, we must carefully select the strains and the environment and conduct preliminary experiments before applying FeOB to As-contaminated paddy fields. CRediT authorship contribution statement Anwen Xiao: Writing - original draft, Investigation, Formal analysis. Wai Chin Li: Conceptualization, Visualization, Supervision, Project administration, Funding acquisition. Zhihong Ye: Resources, Methodology, Writing - review & editing. Declaration of competing interest None. Acknowledgements The work described in this article was supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (EdUHK 28100014), the Faculty of Liberal Arts and Social Sciences (Dean’s Research Fund) of the Education University of Hong Kong, the NSFC-Guangdong United Foundation of China (U1501232), and the National Key Research and Development Program of China (2018YFD0800700). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.110136. References Batista, B.L., Souza, J.M.O., De Souza, S.S., Jr, F.B., 2011. Speciation of arsenic in rice and estimation of daily intake of different arsenic species by Brazilians through rice consumption. J. Hazard Mater. 191, 342–348. Begg, C.B.M., Kirk, G.J.D., Mackenzie, A.F., Neue, H.U., 1994. Root‐induced iron oxidation and pH changes in the lowland rice rhizosphere. New Phytol. 128, 469–477. Ben-Dov, E., Shapiro, O.H., Siboni, N., Kushmaro, A., 2006. Advantage of using inosine at the 3′ termini of 16S rRNA gene universal primers for the study of microbial diversity. Appl. Environ. Microbiol. 72, 6902–6906. Blute, N.K., Brabander, D.J., Hemond, H.F., Sutton, S.R., Newville, M.G., Rivers, M.L., 2004. Arsenic sequestration by ferric iron plaque on cattail roots. Environ. Sci. Technol. 38, 6074–6077. Chen, Z., Zhu, Y.G., Liu, W.J., Meharg, A.A., 2005. Direct evidence showing the effect of root surface iron plaque on arsenite and arsenate uptake into rice (Oryza sativa) roots. New Phytol. 165, 91–97. Chen, X.P., Zhu, Y.G., Hong, M.N., Kappler, A., Xu, Y.X., 2008. Effects of different forms of nitrogen fertilizers on arsenic uptake by rice plants. Environ. Toxicol. Chem. 27, 881–887. Chi, Y.H., Li, F.B., Tam, N.F., Liu, C.P., Ouyang, Y., Qi, X.L., Li, W.C., Ye, Z.H., 2018. Variations in grain cadmium and arsenic concentrations and screening for stable lowaccumulating rice cultivars from multi-environment trials. Sci. Total Environ. 643, 1314–1324. Chun, J., Goodfellow, M., 1995. A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int. J. Syst. Bacteriol. 45, 240–245. Deng, D., Wu, S.C., Wu, F.Y., Deng, H., Wong, M.H., 2010. Effects of root anatomy and Fe plaque on arsenic uptake by rice seedlings grown in solution culture. Environ. Pollut. 158, 2589–2595. Dong, M.F., Feng, R.W., Wang, R.G., Sun, Y., Ding, Y.Z., Xu, Y.M., Fan, Z.L., Guo, J.K., 2016. Inoculation of Fe/Mn-oxidizing bacteria enhances Fe/Mn plaque formation and reduces Cd and as accumulation in rice plant tissues. Plant Soil 404, 75–83.
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World Health Organization, 2014. Codex Alimentarius Commission-Geneva. pp. 14–18. Wu, C., Ye, Z., Shu, W., Zhu, Y., Wong, M., 2011. Arsenic accumulation and speciation in rice are affected by root aeration and variation of genotypes. J. Exp. Bot. 62, 2889–2898. Wu, C., Ye, Z., Li, H., Wu, S., Deng, D., Zhu, Y., Wong, M., 2012. Do radial oxygen loss and external aeration affect iron plaque formation and arsenic accumulation and speciation in rice? J. Exp. Bot. 63, 2961–2970. Zhou, Z.H., Chen, L.F., Min, L.I., Xu, X.P., 2011. Study on Fe2+ removing conditions of an iron-oxidizing bacterium. J. Fujian Normal Univ. (Philos. Soc. Sci. Ed.) 27, 85–180 (in Chinese). Zhuang, P., Lu, H., Li, Z., Zou, B., McBride, M.B., 2014. Multiple exposure and effects assessment of heavy metals in the population near mining area in South China. PLoS One 9, e94484.
heavy metals and metalloids in aquatic and wetland plants. Metall 6, 1789–1800. Ünyayar, S., Çelik, A., Çekiç, F.Ö., Gözel, A., 2006. Cadmium-induced genotoxicity, cytotoxicity and lipid peroxidation in Allium sativum and Vicia faba. Mutagenesis 21, 77–81. Wang, Q., Xiong, D., Zhao, P., Yu, X., Wang, G., 2011. Effect of applying an arsenicresistant and plant growth-promoting rhizobacterium to enhance soil arsenic phytoremediation by Populus deltoides LH05-17. J. Appl. Microbiol. 111, 1065–1074. Wang, X., Ye, Z., Li, B., Huang, L., Meng, M., Shi, J., Jiang, G., 2014. Growing rice aerobically markedly decreases mercury accumulation by reducing both Hg bioavailability and the production of MeHg. Environ. Sci. Technol. 48, 1878–1885. Williams, P.N., Lei, M., Sun, G., Huang, Q., Lu, Y., Deacon, C., Meharg, A.A., Zhu, Y.G., 2009. Occurrence and partitioning of cadmium, arsenic and lead in mine impacted paddy rice: Hunan, China. Environ. Sci. Technol. 43, 637–642.
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Effects of Fe-oxidizing bacteria (FeOB) on iron plaque formation, As concentrations and speciation in rice (Oryza sativa L.)
T
Anwen Xiaoa, Wai Chin Lib,∗,1, Zhihong Yea,∗∗ a b
School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China Department of Science and Environmental Studies, The Education University of Hong Kong, Hong Kong, SAR, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Fe-oxidizing bacteria Rice Arsenic Speciation
Large areas of the paddy fields in South China are contaminated with arsenic (As), which causes serious problems, including high As concentrations in brown rice. Three As-resistant iron-oxidizing bacteria (FeOB) namely, Bacillus sp. T2, Pseudomonas sp. Yangling I4 and Bacillus sp. TF1-3, were isolated and applied to rice grown in different As-contaminated environments to study the effects of FeOB on the As accumulation in rice and clarify the possible mechanisms involved. The results showed that FeOB inoculation significantly decreased the inorganic As concentrations in brown rice grown in pots and paddy fields by 3.7–13.3% and 4.6–12.1%, respectively. FeOB inoculation enhanced the formation of Fe plaque, which sequestered more As on the root surface. Moreover, a significantly lower level of As(III) influx was observed in the rice cultivated with FeOB than in the control. FeOB inoculation also decreased the As concentrations in pore water and the Fe(II)/Fe(III) ratio in rhizosphere soil. The present results suggest that FeOB inoculation decreased the inorganic As concentrations in brown rice by affecting the formation of Fe plaque, As(III) uptake kinetics and rhizosphere soil properties. Based on our results, FeOB inoculation could be considered a useful method to decrease inorganic As concentrations in brown rice grown in As-contaminated paddy fields.
1. Introduction
accounting for 70%–88% of the total As in rice grains (Li et al., 2015; Lin et al., 2015). The predominant As species in agricultural soils are iAs, and there is a higher As(III) bioavailability than other As species in flooded paddy fields, which results in more iAs in rice grains (Du et al., 2019). In other words, compared to the total As, we must pay close attention to the iAs concentration in brown rice. Iron (Fe) plaque is an important barrier for roots of wetland plants and prevent damage inflicted by reductive substances on plants after flooding. Moreover, Fe plaques are also a potential barrier for plants exposed to As (Tripathi et al., 2014). In the present study, much lower As concentrations were measured in Typha root tissues than in the Fe plaque on the root surface. Arsenic was spatially located with Fe, indicating that As was absorbed by Fe in Fe plaque (Blute et al., 2004; Mei et al., 2012). In a hydroponic experiment, Fe plaque on rice root surface functioned as a barrier to As and reduced As accumulation in the root and shoot (Wu et al., 2012). In paddy fields, the formation of Fe plaque on the root surface limits As uptake by rice, and most of the As released from soils was distributed in Fe plaque compared to rice tissues (Syu et al., 2013).
Large areas of paddy fields have been contaminated with arsenic (As) because of mining and other human activities in Asia (Williams et al., 2009). Arsenic exerts very toxic effects on plant growth when its concentrations exceed a threshold (Ünyayar et al., 2006). High levels of As in paddy soils result in higher As concentrations in brown rice (Mei et al., 2009; Zhuang et al., 2014). As the most toxic speciation of As, inorganic As (iAs) [As(III), As(V)] is a non-threshold carcinogen, while organic As is less toxic (Batista et al., 2011). Inorganic As exposure can cause skin, lung, and bladder cancers and may result in non-cancerous respiratory, cardiovascular, metabolism and nervous system diseases (Sanchez et al., 2016). Compared to total As concentration, iAs concentration is a better index to estimate the health risk of As in rice (Kumarathilaka et al., 2019; Lin et al., 2015). The World Health Organization standard of iAs in polished rice grains for adults is 200 μg/kg (World Health Organization, 2014). Moreover, investigations in China have demonstrated that most of the As in the rice grains grown in As-contaminated paddy fields is iAs,
∗
Corresponding author. Corresponding author. E-mail addresses: [email protected] (W.C. Li), [email protected] (Z. Ye). 1 Dr. LI WC will handle correspondence at all stages of refereeing and publication, also post-publication. ∗∗
https://doi.org/10.1016/j.ecoenv.2019.110136 Received 14 August 2019; Received in revised form 24 December 2019; Accepted 24 December 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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citrate, 0.5 g of MgSO4·7H2O, 0.5 g of (NH4)2SO4, 0.5 g of K2HPO4, 0.5 g of NaNO3, 0.2 g of CaCl2, and 1 L of distilled water) (Ghosh et al., 2014; Zhou et al., 2011). After incubation, colonies with varying morphologies were selected from the plates and streaked on the same medium at least five times to purify the clone. The strains were then maintained as glycerol stocks stored at −80 °C (Singh et al., 2016a).
Iron plaque affect the rate of As absorption by the roots. Chen et al. (2005) reported that Fe plaque on the rice root surface increased the rate of As(III) absorption but decrease the rate of As(V) absorption. In another hydroponic experiment, Deng et al. (2010) also found that Fe plaque increased the As(III) absorption rate of rice roots, decreased As (V) absorption rate at low As(V) concentration (< 2 mg/L), but increased As(V) absorption rate at high As(V) concentration (> 6 mg/L). Because some management techniques could induce Fe plaque formation, As accumulation in rice may be reduced. For example, when the amount of added sulfur increases, the As concentration in the rice shoots decreases, which is attributed to the increased formation of Fe plaque on the root surface and in rhizosphere soil (Hu et al., 2007). Previous studies have reported that Fe-oxidizing bacteria (FeOB) promotes the formation of Fe plaque on the roots of wetland plants (Neubauer et al., 2007; Schmidt and Eickhorst, 2013). In a wetland plant (e.g., Juncus effusus) rhizosphere, high densities of lithotrophic FeOB in a favourable environment [such as high Fe(II) availability] suggests that FeOB actively contribute to the formation of Fe plaque on plant roots (Neubauer et al., 2007). The abundance of FeOB in the rhizosphere area correlates with Fe content on the root surface and suggests that FeOB plays important roles in the formation of Fe plaque (Begg et al., 1994; Emerson et al., 1999; Schmidt and Eickhorst, 2013). Moreover, artificial treatment also shows that FeOB inoculation increases the formation of Fe plaque on the surfaces of reed roots (Guo and Cutright, 2015). FeOB have a close relationship with As and Fe levels in soil. In axenic hydroponic microcosms, the presence of FeOB in the J. effusus rhizosphere increases the short-term rates of Fe(II) oxidation by 1.3–1.7 times compared to a control (Neubauer et al., 2007). As shown in the study by Neubauer et al. (2002), biotic oxidation of FeOB competes with abiotic Fe(II) oxidation and accounts for up to 60% of the total Fe (II) oxidation in laboratory cultures. Moreover, the growth of FeOB stimulates nitrate-dependent Fe(II) oxidation, leading to As co-precipitation or adsorption to Fe(III) minerals in the soil, which sequesters the mobile As in the soil and results in a reduced As uptake by rice (Chen et al., 2008). Previous studies show that Fe plaque on rice root surface reduce As accumulation, therefore, FeOB, which promotes the formation of Fe plaque implies that FeOB inoculation may affect As accumulation in rice grown in As-contaminated soils. However, studies on the influence of FeOB on Fe plaque formation and As accumulation in rice grown in As-contaminated environments are rare. Only one study showed that FeOB inoculation increased Fe plaque formation and decreased As accumulation in rice tissues in a pot experiment (Dong et al., 2016). Unfortunately, the mechanisms underlying these phenomena remain unclear, and the effect of FeOB on rice grown in paddy fields is also unknown. A series of experiments (including hydroponic, pot and field experiments) were conducted to answer these questions. The aims of this paper are to investigate the effects of FeOB on As (including iAs) accumulation in rice, elucidate the possible mechanisms involved and explore whether FeOB represents a useful modification to As-contaminated paddy soil.
2.1.1. Arsenic resistance ability All strains were cultivated onto pH 7.0 FeOB media containing 1000 mg/L As(V), placed in a 28 °C incubator shaker for 2 days, and the growth of the strains was determined by measuring the absorbance at 600 nm using the method reported by Hohmann et al. (2010). The FeOB media that were not inoculated with a strain were used as a reference. 2.1.2. Fe oxidation ability All strains were cultivated onto pH 7.0 FeOB media and then placed in a 28 °C incubator shaker for 7 days. The FeOB media from the upper layer was collected and filtered through a 0.45 μm membrane filter (MICROPORE, Minigene Syringe Filter, Genetix Biotech Asia Pvt. Ltd) to remove the particulate material. Phenanthroline photometry was used to determine Fe(II) concentrations (Zhou et al., 2011). Five millilitres of an acetic acid-ammonium acetate buffer solution and a 0.5% 1,10-phenanthroline solution were added to a 50 mL sample, and after 10 min, the absorbance of the mixed solution was determined at 510 nm. Distilled water was used for a reference. 2.1.3. As fixation ability All strains were cultivated onto FeOB media that contained 1 mg/L As(V) and then placed in a 28 °C incubator shaker for 7 days. The FeOB media were filtered, and the As concentration was determined using atomic fluorescence spectrometry (AFS, Beijing Titan Instrument Co. Ltd.) (Hohmann et al., 2010). 2.1.4. Indole acetic acid (IAA) This experiment was performed using the methods described by Mahmood et al. (2017). Bacterial cultures were grown in a Pikovskaya medium with tricalcium phosphate under standard conditions for 120 h to determine the solubilization of phosphates (Iyer et al., 2017). The supernatants from the cultures were centrifuged at 12,857×g for 10 min. The concentration of soluble phosphate in the supernatant was estimated using the phosphomolybdate blue method (Islam et al., 2014). 2.1.5. Identification by 16S rRNA gene sequencing The above strains that were identified with a relatively higher tolerance were selected for genetic identification. Genomic DNA was extracted, and the 16S rRNA gene was amplified by PCR using the procedures described by Chun and Goodfellow (1995). The universal primers for the bacterial 16S rRNA gene (the forward primer P1 5′-CGG GAT CCA GAG TTT GAT CCT GGC TCA GAA CGA ACG CT- 3′ and the reverse primer P6 5′-CGG GAT CCT ACG GCT ACC TTG TTA CGA CTT CAC CCC-3′) were used to amplify the target gene (Ben-Dov et al., 2006). The PCR product was purified and directly sequenced with an automated DNA Sequencing System (ABI 3730XL). Almost complete 16S rRNA gene sequences were obtained and matched, using the BLASTn program (http://blast.ncbi.nlm.nih.gov/), to nucleotide sequences in the GenBank, European Molecular Biology Laboratory (EMBL) and the DNA Data Bank of Japan (DDBJ) databases.
2. Materials and methods 2.1. FeOB isolation, identification and characterization The rice rhizosphere soil samples were collected from an As-contaminated paddy field near the Baoshan mine (112°44′18.64″E, 25°43′4.38″N), Chenzhou City, Hunan Province, China, which has been described by Liao et al. (2005). The soil samples (1 g) were suspended in 50 mL of a modified Wolfe mineral media at a pH of 6.0 and incubated at 37 °C for 30 min (Ghosh et al., 2014). The supernatant was filtered through a 0.45 μm membrane filter (MICROPORE, Minigene Syringe Filter, Genetix Biotech Asia Pvt. Ltd). For isolating FeOB, the filtrate was plated onto pH 7.0 FeOB media (10 g of ferric ammonium
2.2. Inoculum and seedling preparation The selected rhizobacterial isolates were grown in FeOB medium at 28 °C for 1 day. Bacterial cells were harvested by centrifugation and resuspended in distilled water prior to inoculation. The rice cultivar Jianyou G2 (which is widely cultivated in Guangdong Province) was chosen, and the seeds were purchased from 2
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Golden Rice Seeds Co. Ltd. of Guangdong. Seeds were surface sterilized with a 30% H2O2 solution for 15 min and washed with deionized water. For hydroponic and pot experiments, the seeds were germinated in sterilized petri dishes containing moist filter paper in a controlled chamber (28 °C and 70% relative humidity) (Wu et al., 2011). 2.3. Analysis of the uptake kinetics The study of As uptake kinetics in roots was performed using the method described by Li et al. (2011). Three-week-old rice seedlings were incubated with (i) a bacterial suspension in sterile distilled water (109 cfu/mL) or (ii) sterile distilled water for 1 h as control (Dong et al., 2016; Singh et al., 2016a, 2016b). After different treatments, the seedlings were excised, and then the roots were incubated for 40 min with test solutions that contained arsenite (NaAsO2) or arsenate (Na2HAsO4·7H2O). All test solutions contained 5.0 mM 2-(N-morpholino)-ethanesulfonic acid (MES) and 0.5 mM Ca(NO3)2, with the pH adjusted to 5 using KOH. Next to the roots were rinsed for 15 min with an ice-cold phosphate solution that contained 1 mM K2HPO4, 5 mM MES and 0.5 mM Ca(NO3)2 to remove the absorbed As species from the root free space. Finally, the roots were washed and oven-dried at 60 °C and then the As concentrations in the root tissues were analysed. 2.4. Hydroponic experiment Uniform rice seedlings were transplanted to plastic vessels and incubated with a ¼ dilution of Hoagland's nutrient solution (Hoagland μmol NaHAsO•7HO for 30 d and Arnon, 1938) and 40 . The nutrient solutions 2 4 2 were replaced once every 3 days during the growth period. All vessels were arranged randomly in a controlled greenhouse with natural light supplemented by sodium light and a day/night temperature of 25/ 20 °C, a day/night photoperiod of 12/12 h and a relative humidity of 70%. Seedlings of each cultivar were incubated with (i) a bacterial suspension in sterile distilled water (109 cfu/mL) or (ii) sterile distilled water for 1 h as control (Dong et al., 2016; Singh et al., 2016a, 2016b). Four replicates were prepared for each treatment. After 36 days, the plants were carefully washed with deionized water and separated into shoots and roots. All samples were freeze-dried and milled to a powder for concentration analyses of the total As and As species [arsenite (As (III)), arsenate (As(V)), dimethylarsinate (DMA), and monomethylarsonate (MMA)].
Fig. 1. Grain yield of rice grown in pots with As-contaminated soils collected from Dabaoshan (a) and paddy fields at Dabaoshan (b) with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments within the same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
2.5. Pot experiment
2016b). Pore water was collected at the rice heading stage using rhizon soil pore water samplers (Rhizosphere Research Products, The Netherlands), as previously reported by Kidd et al. (2007). Rice plants were harvested at the mature stage and separated into roots, straw and brown rice. All plant samples (except roots) were washed, freeze-dried at −50 °C, milled to a fine powder, and stored at 4 °C until further analysis. Roots were divided into two subsamples: one for the enzyme analysis and another for the total As analysis. The total concentrations of As in rice tissues and pore water were analysed using AFS after microwave digestion. The concentrations of As species in the rice were determined by weighing 0.4 g of rice flour into a 10 mL polypropylene tube and extracting the sample with 8 mL of 1% HNO3 at 90 °C for 2.5 h (Lin et al., 2015). The extract was then centrifuged at 8228×g for 10 min at 4 °C, and the supernatant was filtered
For the pot experiment, the soil was collected from the surface (0–20 cm) of two As-contaminated paddy fields in Dabaoshan (DBS, 113°49′5.6″E, 24°28′44.1″N), Shaoguan City, Guangdong Province, and named DA and DB, respectively. The basic soil properties are shown in Table 1. A cylindrical nylon rhizobag (30 μm nylon mesh, 12 cm in diameter and 15 cm in height) was designed to separate the rhizosphere soil from the bulk soil (Wang et al., 2014). Uniform rice seedlings were carefully transplanted into each rhizobag. Four replicates were prepared for each treatment (Wang et al., 2014). FeOB (as a bacterial suspension in sterile distilled water, 109 cfu/mL) and sterile distilled water (control) were applied to the rhizobag three times (at the transplanting, tillering and heading stages, respectively) (Dong et al., 2016; Singh et al., 2016a, Table 1 Basic properties of soils collected from the two paddy fields (Mean ± SE, n = 6).
DA DB
As (mg/kg)
Mn (mg/kg)
Zn (mg/kg)
Cu(mg/kg)
Fe (g/kg)
K (g/kg)
P (g/kg)
pH
138 ± 8.7 41 ± 1.9
153 ± 8.2 178 ± 5.0
320 ± 19 292 ± 17
370 ± 20 408 ± 26
38 ± 2.2 29 ± 0.4
28 ± 1.1 26 ± 2.0
11 ± 0.83 12 ± 0.51
5.22 ± 0.21 5.47 ± 0.18
Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. 3
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Fig. 2. Arsenic concentration in brown rice grown in pots with DA (a) and DB (b) soil and in DA (c) and DB (d) paddy fields and DMA concentration in brown rice grown in pots (e) and fields (f) with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments within the same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3. 4
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Fig. 3. The concentration of As in shoots (a) and roots (b) in pot experiments with different FeOB strains inoculation and As concentration in shoots (c) and roots (d) in field experiments with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments within the same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
through a 0.22 μm polyethersulfone membrane (Tianjin Branch Billion Lung Experimental Equipment Co. Ltd. Tianjin, China). The samples were stored at 4 °C and analysed within a few hours after extraction to minimize the transformation of As species. Arsenic speciation in the rice extracts was performed using atomic fluorescence spectrometry-highperformance liquid chromatography (AFS-HPLC, Beijing Titan Instrument Co. Ltd.) (Chi et al., 2018). The dithionite-citrate-bicarbonate (DCB) method (Otte et al., 1991; Taylor and Crowder, 1983) was used to extract Fe plaque on the fresh root surfaces of rice plants. Fresh roots were extracted for 3 h in a solution of 30 mL of 0.03 M Na3C6H5O7·2H2O, 0.125 M NaHCO3 and 0.6 g of Na2S2O4 at room temperature (25 °C). After extraction, the roots were washed 3 times with 15 mL of deionized water, and the wash was collected. The volumes of the resulting extracts were brought to 50 mL with deionized water, and then the concentrations of Fe and As were analysed. The soil within the rhizobags was regarded as the rhizosphere soil (Wang et al., 2014). The rhizosphere soil samples were freeze-dried at −50 °C, crushed, sieved through a 150 mesh, and collected to measure the levels of extractable As, the pH and the soluble P. The pH was measured with a glass electrode (soil: water = 1:2.5) (Lu, 1999). The As concentrations from the soil extract were measured using AFS after a 2 h extraction in leach liquor (0.01 M CaCl2, solution: soil = 5:1) (Li et al., 2014). The Fe(II)/Fe(III) ratio was calculated using the method reported by Begg et al. (1994). The rice rhizosphere soil was collected and approx. 1 g of soil was placed in a tube containing 3 mL of 5 mM CaCl2 under an anaerobic environment. After the addition 10 mL of 1 M NH4OAc (pH
2.8), each tube was sealed and shaken for 30 min. The extracts were centrifuged at 1575×g for 10 min. The supernatant was filtered through a 0.45 μm membrane filter and used for the Fe(II) assay. Ten millilitres of 2 M H2SO4 was added to the residue, and then the tube was shaken for 30 min. The extracts were centrifuged at 1575×g for 10 min and the filtered supernatant was used for the Fe(III) assay. 2.6. Field experiments Field experiments were conducted at two paddy fields in Dabaoshan, as mentioned above. The rice cultivar Jianyou G2 was chosen. Each treatment included four replicates with a 3 m × 3 m plot (Wang et al., 2014). All plots were randomly located in each paddy field. Two rice seedlings were carefully transplanted into each rhizobag. FeOB inoculation and control conditions were similar to the pot experiment. Field management was performed according to the strategies employed locally, except for the addition of FeOB. Rice samples were harvested at the mature stage, and the sample treatment methods were similar to the pot experiment. 2.7. Quality control and statistical analyses Blanks, soil standard material (GBW-07401), plant standard material (GBW-07435) and brown rice standard material (GBW-10013) (China Standard Materials Research Centre, Beijing, P.R. China) were used for quality control. Recoveries from the reference materials ranged from 85.1% to 92.0% for the total As analysis and 87.0%–91.2% for the iAs. 5
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Fig. 4. The concentration of Fe (a) and As (b) in Fe plaque on root surface in pot experiments with different FeOB strains inoculation and the concentration of Fe (c) and As (d) in Fe plaque on root surface in field experiments with different FeOB strains inoculation (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A and Dabaoshan-B, respectively. Data with different letters mean significant differences between the treatments in same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
In the hydroponic experiment, the As concentration in the shoots of rice with F1 inoculation was reduced by 42.6%–63.5% (Fig. S6a). The total As concentration in the roots was significantly decreased by FeOB (P < 0.05), and the As(III) and As(V) concentrations were also obviously decreased by 10.8%–36.4%, after FeOB inoculation (Fig. S6b). Interestingly, the roots of rice grown in hydroponic culture contained only iAs, while the shoots contained As(III), As(V) and DMA (Fig. S6). Compared with the control, the total As concentration in brown rice with F3 and F10 inoculation was significantly reduced by 15.7% and 18.9%, respectively, in pots with DA soil (Fig. S7a). As for As species, the FeOB (except F3) significantly decreased the iAs concentration in brown rice grown in pots by 3.7%–13.3% (P < 0.05), which was attributed to the decrease in As(III) concentration by 4.0%–16.3% (Fig. 2a and b). Regarding the organic As concentration in brown rice, FeOB inoculation significantly decreased the DMA concentration in brown rice grown in pots with DA soil by 18.9%–36.0% (P < 0.05) (Fig. 2e). In DB paddy field experiment, total As in brown rice with F1 and F10 inoculation also significantly decreased by 11.4% and 17.5%, respectively (Fig. S7b). The FeOB did not affected As concentration in brown rice grown at DA paddy field, however, the FeOB could significantly decreased the iAs concentration in brown rice by 8.4%–12.1% under DB field conditions (P < 0.05) (Fig. 2c and d). In field experiments, F1 and F10 inoculation significantly decreased the DMA concentration in brown rice grown at DB by 21.4% and 36.1%, respectively (P < 0.05) (Fig. 2f). In pot experiments, FeOB inoculation significantly increased As concentrations in rice shoots and roots by 13.3%–137.4% and 11.9%–57.8%, respectively (P < 0.05) (Fig. 3a and b). However, the
Origin 8.0 and Excel 2007 software were used to create the artwork. Data were analysed using the SPSS 19.0 statistical software package and summarized as the means ± standard error (SE). Treatment means were compared using the Duncan multiple comparisons test at the 5% level of significance. 3. Results 3.1. The isolated FeOB Twelve FeOB were isolated from the rhizosphere soil of rice grown in an As-contaminated paddy field. Based on their Fe oxidation ability, As resistance, As fixation ability, phosphate solubilizing and IAA production capacity, three FeOB (F1, F3, and F10) were selected, and some of their properties are shown in Figs. S1–S5. Compared to the other strains, the three selected FeOB all exhibited a higher As resistance, Fe oxidation ability and As fixation ability (Figs. S1–S3). The results from physiological and biochemical analyses and 16S rDNA sequencing showed that F1 was Bacillus sp. T2, F3 was Pseudomonas sp. Yangling I4 and F10 was Bacillus sp. TF1-3. 3.2. The effect of FeOB on the grain yield and As accumulation in rice The grain yield with F1 and F10 inoculation increased by 40.1% and 25.6%, respectively, in pots with the DB soil (Fig. 1a). However, the grain yield of rice grown in pots with DA soil and in two paddy fields did not differ significantly between the plants with and without FeOB inoculation (Fig. 1b). 6
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particularly in roots cultured with F3. 3.4. Soil properties In the pot experiments, compared to the control, the Fe(II)/Fe(III) ratio in rhizosphere soil of rice plant inoculated with FeOB decreased, particularly for the plant inoculated with F1 and F10, which significantly decreased the Fe(II)/Fe(III) ratio by 7.7%–44.2% (P < 0.05), except for F1 in DA (Fig. 6a). The FeOB did not substantially alter the rhizosphere soil pH or the available As concentration in soils of the pot trials (Fig. 6b and c). However, following F1 and F10 inoculation, As concentrations in soil pore water were significantly reduced by 26.7%–55.0% (P < 0.05), except for the DA inoculated with F10 (Fig. 6d). 4. Discussion According to the 16S rRNA gene sequencing data, the three isolated FeOB belong to Pseudomonas (F3) and Bacillus (F1 and F10), respectively. These results were consistent with a recent study indicating that some FeOB belong to these two genera (Ghosh et al., 2014). 4.1. As concentration in rice plants Compared to the total As, the iAs concentration in brown rice requires more attention because its toxicity is higher than that of other As species, and iAs is a better index than total As (Batista et al., 2011; Kumarathilaka et al., 2019; Lin et al., 2015). In the present study, iAs concentration in brown rice decreased following FeOB inoculation in all hydroponic, pot and field experiments, particularly after the inoculation of F1 and F10 (Fig. 2 and S6), indicating that FeOB inoculation could effectively reduce the risk of food safety hazards from rice grown in As-contaminated paddy fields. In a previous study, Dong et al. (2016) reported that the total As concentration in brown rice decreased following FeOB inoculation in a pot experiment. The explanations for this decrease are related to the changes in soil properties and the rice plants following FeOB inoculation. On the one hand, As concentrations in soil pore water were significantly decreased following the F1 or F10 treatment, which may be due to the decrease in the Fe(II)/Fe(III) ratio in the rhizosphere soil inoculated with F1 and F10 (Fig. 6). Consistent with the present results, some previous studies also found that FeOB increased Fe(II) oxidation rates in soil and that the growth of FeOB sequestered more mobile As due to increased Fe(III) formation (Chen et al., 2008; Neubauer et al., 2007). All three selected strains decreased mobile As and Fe concentrations in FeOB media (Figs. S2 and S3). On the other hand, compared to the control, significantly lower As (III) influx was observed following FeOB inoculation (P < 0.05) (Fig. 5a), which was consistent with the decrease in As(III) concentrations in brown rice (Fig. 2). In addition, in DA, the concentrations of Fe and As in Fe plaque increased with FeOB inoculation (Fig. 4), indicating that FeOB inoculation promoted Fe plaque formation and then fixed more As in the Fe plaque, which contributed to the decrease of iAs concentration in the brown rice. A previous study reported that FeOB promotes Fe plaque formation on the roots of wetland plants (Neubauer et al., 2007), which was consistent with the results of the present study (Fig. 4). The increased formation of Fe plaque on the root surface may account for the decrease in As(III) uptake by rice roots cultured with FeOB. Iron plaque affect As uptake by plant roots, and some of the As is fixed in the Fe plaque, reducing the As accumulation in plant tissues (Blute et al., 2004; Deng et al., 2010; Syu et al., 2013). Moreover, the decreased rate of As transfer in rice tissues may contribute to the decrease of As concentrations in brown rice, particularly in the DA soil (Table S1). The effects of FeOB inoculation on As concentration in shoot and root tissues were different between the rice grown in the pots and in the
Fig. 5. Concentration-dependent kinetics for As(III) (a) and As(V) (b) uptake by rice roots with different FeOB strain suspension. (Mean ± SE, n = 4). Note: CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
As concentrations in the shoots and roots of rice grown in paddy fields inoculated with FeOB (F1 and F10) did not significantly differ from the control (Fig. 3c and d). 3.3. The Fe plaque and As uptake kinetics of rice roots In pot experiments, FeOB inoculation increased Fe concentrations in Fe plaque on root surfaces and a significant increase was noticed in DA (11%–28.1%) (P < 0.05) (Fig. 4a). Moreover, the As concentrations in Fe plaque also increased following FeOB inoculation by 14.6%–42.5%, except for the inoculation of F1 in DB (Fig. 4b). In both DA and DB paddy field experiments, following FeOB (F1 and F10) inoculation, concentrations of Fe and As in Fe plaque were significantly increased by 42.5–50.8% and by 46.1–66.6%, respectively (P < 0.05) (Fig. 4c and d). A significantly lower As(III) influx (decreased by 37.7%–74.6%) was observed in rice roots incubated with FeOB than in roots without FeOB (P < 0.05) (Fig. 5a). The influx of As(V), for which the external concentrations ranged from 0.02 to 0.12 mM, in roots cultured with FeOB was significantly higher than in the control (Fig. 5b) (P < 0.05), 7
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Fig. 6. Fe(II)/Fe(III) (a), pH(b), extracted As concentration(c) and As concentration in pore water(d) in rhizosphere soil of rice with different FeOB strains inoculation in pot experiment. (Mean ± SE, n = 4). Note: DA and DB represent Dabaoshan-A soil and Dabaoshan-B soil, respectively. Data with different letters mean significant differences between the treatments in same site at 0.05 levels. CK: control; F1, F3 and F10 were selected FeOB. F1: Bacillus sp. T2, F3: Pseudomonas sp. Yangling I4, F10: Bacillus sp. TF1-3.
paddy fields. FeOB inoculation increased As concentrations in the shoots and roots of rice in the pot trials; however, the results were different in the paddy field experiments (Fig. 3). A potential explanation was that rice grown in pots absorbed more As than rice grown in fields (Figs. 2 and 3), which altered the effect of the FeOB.
to the plant growth-promoting traits of FeOB (Fig. 5b, S4 and S5). Interestingly, although FeOB increased As(V) influx in the rice roots, FeOB did not increase As(V) concentrations in the brown rice. A potential explanation for this finding was the presence of Fe plaque on the root surface and the As distribution in rice tissues. The increased As(V) influx may be mainly sequestered in Fe plaque on the root surface and in the rice roots and shoots (Figs. 3 and 4).
4.2. Rice grain yields Compared with the control, F1 and F10 inoculation significantly increased the grain yield of the rice grown in pots with DB soil (Fig. 1). The results suggested that some FeOB (e.g., F1 and F10) have some plant growth-promoting traits (e.g., phosphate solubilization and the production of IAA-like molecules) (Figs. S4 and S5). Thus, these bacteria may be a combination of FeOB and plant growth-promoting rhizobacteria, which decrease the iAs concentration in brown rice and increase the grain yield of rice grown in As-contaminated paddy fields. However, the grain yields were not significantly different between the treatments with and without FeOB inoculation in the pots with DA soil and in the paddy fields. These results suggested that the effects of FeOB on grain yields may also be regulated by other environmental factors (e.g., soil types).
4.4. The importance of environmental factors The present results showed that the effect of FeOB inoculation on As concentrations in rice tissues differed in different environments. Compared to DA, the effect of FeOB inoculation on plants grown in DB soil was more obvious, which may be attributed to the differences in soil properties, particularly the total As concentration in soil (Fig. 6 and Table 1), as the total As concentration in DB soil was significantly lower than in DA soil (P < 0.05). Based on the results from the hydroponic, pot and field experiments (Fig. 2 and S6), the difference of As concentration in rice between control and rice inoculated with FeOB was reduced. The number of influencing factors increased when the experimental environment changed from the hydroponic systems and pots to the paddy fields, making the variables more difficult to control. Moreover, during strain inoculation, inoculation in a field would dilute the influence of FeOB on rice and soil because of the abundant microbial communities in the soil compared to the pot and hydroponic experiments, which would result in a lower effect of the FeOB on the rice grown in paddy fields. Notably, researchers must be conscious of the different behaviours of FeOB in different environments and conduct
4.3. The relationship between the As(V) influx and FeOB traits Wang et al. (2011) reported that IAA stimulated the elongation of cells or increased cell division in plant roots, which resulted in increased As absorption in the roots. Therefore, compared to the control, the higher root As(V) influx in rice cultivated with FeOB may be related 8
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preliminary experiments before applying FeOB in As-contaminated paddy fields to reduce As risks.
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5. Conclusions In the present study, FeOB decreased As accumulation in brown rice in both pot and paddy field trials without an adverse effect on the grain yield, indicating that the application of a suitable FeOB is possible to mitigate the problem of As-contaminated brown rice after carefully selecting the appropriate strains and environment. Because the effects of FeOB were influenced by both the strains and environmental factors, we must carefully select the strains and the environment and conduct preliminary experiments before applying FeOB to As-contaminated paddy fields. CRediT authorship contribution statement Anwen Xiao: Writing - original draft, Investigation, Formal analysis. Wai Chin Li: Conceptualization, Visualization, Supervision, Project administration, Funding acquisition. Zhihong Ye: Resources, Methodology, Writing - review & editing. Declaration of competing interest None. Acknowledgements The work described in this article was supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (EdUHK 28100014), the Faculty of Liberal Arts and Social Sciences (Dean’s Research Fund) of the Education University of Hong Kong, the NSFC-Guangdong United Foundation of China (U1501232), and the National Key Research and Development Program of China (2018YFD0800700). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.110136. References Batista, B.L., Souza, J.M.O., De Souza, S.S., Jr, F.B., 2011. Speciation of arsenic in rice and estimation of daily intake of different arsenic species by Brazilians through rice consumption. J. Hazard Mater. 191, 342–348. Begg, C.B.M., Kirk, G.J.D., Mackenzie, A.F., Neue, H.U., 1994. Root‐induced iron oxidation and pH changes in the lowland rice rhizosphere. New Phytol. 128, 469–477. Ben-Dov, E., Shapiro, O.H., Siboni, N., Kushmaro, A., 2006. Advantage of using inosine at the 3′ termini of 16S rRNA gene universal primers for the study of microbial diversity. Appl. Environ. Microbiol. 72, 6902–6906. Blute, N.K., Brabander, D.J., Hemond, H.F., Sutton, S.R., Newville, M.G., Rivers, M.L., 2004. Arsenic sequestration by ferric iron plaque on cattail roots. Environ. Sci. Technol. 38, 6074–6077. Chen, Z., Zhu, Y.G., Liu, W.J., Meharg, A.A., 2005. Direct evidence showing the effect of root surface iron plaque on arsenite and arsenate uptake into rice (Oryza sativa) roots. New Phytol. 165, 91–97. Chen, X.P., Zhu, Y.G., Hong, M.N., Kappler, A., Xu, Y.X., 2008. Effects of different forms of nitrogen fertilizers on arsenic uptake by rice plants. Environ. Toxicol. Chem. 27, 881–887. Chi, Y.H., Li, F.B., Tam, N.F., Liu, C.P., Ouyang, Y., Qi, X.L., Li, W.C., Ye, Z.H., 2018. Variations in grain cadmium and arsenic concentrations and screening for stable lowaccumulating rice cultivars from multi-environment trials. Sci. Total Environ. 643, 1314–1324. Chun, J., Goodfellow, M., 1995. A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int. J. Syst. Bacteriol. 45, 240–245. Deng, D., Wu, S.C., Wu, F.Y., Deng, H., Wong, M.H., 2010. Effects of root anatomy and Fe plaque on arsenic uptake by rice seedlings grown in solution culture. Environ. Pollut. 158, 2589–2595. Dong, M.F., Feng, R.W., Wang, R.G., Sun, Y., Ding, Y.Z., Xu, Y.M., Fan, Z.L., Guo, J.K., 2016. Inoculation of Fe/Mn-oxidizing bacteria enhances Fe/Mn plaque formation and reduces Cd and as accumulation in rice plant tissues. Plant Soil 404, 75–83.
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