Applied Microbiology and Biotechnology Influence of water management on the active root-associated microbiota involved in arsenic, iron and sulfur cycles in rice paddies --Manuscript Draft-- Manuscript Number: AMAB-D-17-00287R2 Full Title: Influence of water management on the active root-associated microbiota involved in arsenic, iron and sulfur cycles in rice paddies Article Type: Original Article Section/Category: Environmental biotechnology Corresponding Author: Lucia Cavalca Universita degli Studi di Milano ITALY Corresponding Author Secondary Information: Corresponding Author's Institution: Universita degli Studi di Milano Corresponding Author's Secondary Institution: First Author: Sarah Zecchin, PhD First Author Secondary Information: Order of Authors: Sarah Zecchin, PhD Anna Corsini, PhD Maria Martin, PhD Lucia Cavalca Order of Authors Secondary Information: Funding Information: Ministero dell’Istruzione, dell’Università e Dr. Sarah Zecchin della Ricerca (IT) Ministero dell’Istruzione, dell’Università e Not applicable della Ricerca (PRIN 2010JBNLJ7-004) Abstract: In recent years the role of microorganisms inhabiting rice rhizosphere in promoting arsenic contamination has emerged. However, little is known concerning the species and metabolic properties involved in this phenomenon. In this study, the influence of water management on the rhizosphere microbiota in relation to arsenic dissolution in soil solution was tested. Rice plants were cultivated in macrocosms under different water regimes: continuous flooding, continuous flooding with a 2 weeks-period drainage before flowering and dry soil watered every 10 days. The active bacterial communities in rhizosphere soil and in rhizoplane were characterized by 16S rRNA pyrosequencing. An in-depth analysis of microbial taxa with direct or indirect effects on arsenic speciation was performed and related contribution was evaluated. Continuous flooding promoted high diversity in the rhizosphere, with the plant strongly determining species richness and evenness. On the contrary, under watering the communities were uniform, with little differences between rhizosphere soil and rhizoplane. Arsenic-releasing and arsenite-methylating bacteria were selected by continuous flooding, where they represented 8 % of the total. On the contrary, bacteria decreasing arsenic solubility were more abundant under watering, with relative abundance of 10 %. These values reflected arsenic concentrations in soil solution, respectively 135 µg L-1 and negligible in continuous flooding and under watering. When short-term drainage was applied before flowering, intermediate conditions were achieved. This evidence strongly indicates an active role of the rhizosphere microbiota in driving arsenic biogeochemistry in rice paddies, influenced by water management, explaining Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Click here to download Manuscript Zecchin_et_al_AMB_Unmarked_Revision2.doc Click here to view linked References 1 Influence of water management on the active root-associated microbiota 1 2 involved in arsenic, iron and sulfur cycles in rice paddies 2 a a b a* 3 3 Sarah Zecchin , Anna Corsini , Maria Martin and Lucia Cavalca 4 5 4 6 5 7 8 6 aDipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente (DeFENS), Università degli 9 10 7 Studi di Milano, Milano, Italy 11 12 8 13 b 14 9 Department of Agriculture, Forest and Food Science, University of Turin, Turin, Italy 15 16 10 17 11 18 19 12 * Corresponding author 20 21 13 E-mail address: [email protected] 22 23 14 Telephone: +39 02 503 19116 24 25 15 Fax: +39 02 503 19238 26 27 16 28 17 29 30 18 Authors E-mail addresses 31 32 19 [email protected], [email protected], [email protected] 33 34 20 35 36 21 37 22 38 39 23 40 41 24 42 43 25 44 45 26 46 47 27 48 28 49 50 29 51 52 30 53 54 31 55 56 32 57 58 33 59 34 60 61 35 62 63 1 64 65 36 Abstract 1 37 2 38 In recent years, the role of microorganisms inhabiting rice rhizosphere in promoting arsenic 3 4 39 contamination has emerged. However, little is known concerning the species and metabolic 5 6 40 properties involved in this phenomenon. In this study, the influence of water management on the 7 8 41 rhizosphere microbiota in relation to arsenic dissolution in soil solution was tested. 9 10 42 Rice plants were cultivated in macrocosms under different water regimes: continuous flooding, 11 12 43 continuous flooding with a 2 weeks-period drainage before flowering and dry soil watered every 10 13 44 days. The active bacterial communities in rhizosphere soil and in rhizoplane were characterized by 14 15 45 16S rRNA pyrosequencing. An in-depth analysis of microbial taxa with direct or indirect effects on 16 17 46 arsenic speciation was performed and related contribution was evaluated. 18 19 47 Continuous flooding promoted high diversity in the rhizosphere, with the plant strongly determining 20 21 48 species richness and evenness. On the contrary, under watering the communities were uniform, with 22 23 49 little differences between rhizosphere soil and rhizoplane. Arsenic-releasing and arsenite- 24 50 methylating bacteria were selected by continuous flooding, where they represented 8 % of the total. 25 26 51 On the contrary, bacteria decreasing arsenic solubility were more abundant under watering, with 27 28 52 relative abundance of 10 %. These values reflected arsenic concentrations in soil solution, 29 30 53 respectively 135 µg L-1 and negligible in continuous flooding and under watering. When short-term 31 32 54 drainage was applied before flowering, intermediate conditions were achieved. 33 34 55 This evidence strongly indicates an active role of the rhizosphere microbiota in driving arsenic 35 56 biogeochemistry in rice paddies, influenced by water management, explaining amounts and 36 37 57 speciation of arsenic often found in rice grains. 38 39 58 40 41 59 42 43 60 44 61 45 46 47 62 Keywords 48 63 Rice rhizosphere microbiota; arsenic bacteria; sulfate-reducing bacteria; iron-reducing bacteria; 49 50 64 sulfur-oxidizing bacteria; iron-oxidizing bacteria. 51 52 65 53 54 66 55 56 67 57 58 68 59 69 60 61 70 62 63 2 64 65 71 Introduction 1 72 2 73 Arsenic contamination of groundwater resources and soils represents an issue in many areas of the 3 4 74 world (Singh et al. 2015; Heikens 2006). However, arsenic (As) speciation and the physicochemical 5 6 75 characteristics of the environment determine its bioavailability more than its concentrations. In rice 7 8 76 fields the prolonged flooding usually adopted for cultivation leads to As release from soil minerals 9 10 77 with the consequent accumulation of the metalloid in the grains (Zhu et al. 2014; Sun et al. 2014; 11 12 78 Ma et al. 2014). Recent studies revealed that, on average, As content in rice from different countries 13 79 exceeds the law limits established by the Commission regulation (EU) 2015/1006 (Ma et al. 2014; 14 15 80 EFSA 2009, 2014). 16 17 81 The two inorganic As species mainly present in rice field soil, arsenate [As(V)] and arsenite 18 19 82 [As(III)], have different biogeochemical properties. Nevertheless, both As species show high 20 21 83 affinity for iron oxides/hydroxides (Meharg 2012; Martin et al. 2014). 22 23 84 Continuous flooding in paddy soils leads to strongly reduced conditions, with the consequent rapid 24 85 dissolution of these minerals and the release of As into the pore water (Zhu et al. 2014; Meharg 25 26 86 2012). Furthermore, As(V) is reduced to As(III) abiotically by sulfide, ferrous iron [Fe(II)], H2 or 27 28 87 reduced organic acids, or by As(V)-reducing bacteria (Cavalca et al. 2013; Meharg et al. 2012). 29 30 88 Several studies reported that As(III) in flooded rice fields is the predominant As species (Zhu et al. 31 32 89 2014; Takahashi et al. 2004). At very low redox potentials, where microbial sulfate reduction is 33 34 90 favored, sulfide produced by this activity can co-precipitate with As(III) forming a variety of 35 91 minerals, such as orpiment (As2S3) (Zhu et al. 2014; Kocar and Fendorf 2009). If soil is aerated, for 36 37 92 example after a drainage period, As(III) can be oxidized to As(V) by oxygen, manganese oxides 38 39 93 and H2O2 as well as by microbial As(III) oxidation (Meharg 2012). 40 41 94 The genetic properties and the encoded enzymatic systems that allow several groups of 42 43 95 microorganisms to resist to high As concentrations or to use As for metabolic purposes have been 44 96 45 recently reviewed by various authors (Andres et al. 2016; Zhu et al. 2014; Yamamura et al. 2014; 46 97 Cavalca et al. 2013; Zheng et al. 2012; Slyemi and Bonnefoy 2012). Interestingly, in rice paddy 47 48 98 soils with low As concentration a high diversity of microbial genes for As processing has been 49 50 99 detected (Xiao et al. 2016), indicating the potential role of native communities on As 51 52100 transformations beyond abiotic factors. Among these processes, the microbial methylation of 53 54101 As(III) in rice rhizosphere is receiving great attention in the last few years. Recent studies indicated 55 102 that rice roots microbiome is entirely responsible for the production of methylated As present in rice 56 57103 grains, which accounts for 50 % of total As content (Lomax et al. 2012; Arao et al. 2011; Zhao et al. 58 59104 2013). Furthermore, continuous flooding of rice fields has been demonstrated to increase the 60 61105 concentration of methylated As in rice grains (Ma et al. 2014; Li et al. 2009). 62 63 3 64 65 106 In addition to direct As transformations, several metabolic activities of microorganisms could 107 indirectly influence As speciation and bioavailability in the environment.