Arsenite-oxidizing bacteria exhibiting plant growth promoting traits isolated from the rhizosphere of Oryza sativa L.: Implications for mitigation of arsenic contamination in paddies

Highlights

• As(III)-oxidizing bacteria possessing plant growth promoting (PGP) traits isolated.

• The strain produced siderophores, IAA, ACC deaminase and solubilized phosphate.

• Arenite-oxidase (aoxB) gene of the strain was identified.

• Based on 16S rRNA the strain was identified as Bacillus flexus.

• Bacterial inoculation significantly decreased As uptake and increased growth of rice in As-contaminated soil

Abstract

Arsenite-oxidizing bacteria exhibiting plant growth promoting (PGP) traits can have the advantages of reducing As-uptake by rice and promoting plant growth in As-stressed soil. A gram-positive bacterium Bacillus flexus ASO-6 resistant to high levels of As (32 and 280 mM for arsenite and arsenate, respectively) and exhibiting elevated rates of As(III) oxidation (V_max = 1.34 μM min⁻¹ 10⁻⁷ cell) was isolated from rhizosphere of rice. The presence of aoxB gene and exhibition of As(III)-oxidase enzyme activity of this strain was observed. The ability of the strain to produce siderophore, IAA, ACC-deaminase and to solubilize phosphate was verified. The rice seed treated with the strain exhibited significantly improved seed germination and seedling vigor compared with the un-inoculated seeds. The bacterial inoculation significantly increased root biomass, straw yield, grain yield, chlorophyll and carotenoid in the rice plant. Moreover, As uptake from root to shoot and As accumulation in straw and grain decreased significantly as a result of the bacterial inoculation. Noteworthy, the inoculation effect is more prominent in non-flooded soil than it is in flooded soil. Owing to its wide action spectrum, this As(III)-oxidizing PGPB could serve as a potential bio-inoculant for mitigation of As in paddies and sustainable rice production in As-contaminated areas.

Introduction

The indiscriminate use of arsenic (As)-contaminated groundwater for irrigation of agricultural land is a pressing issue of current public health globally. This is because the incorporation of this toxic metalloid into the food chain poses long term risks to human health [1]. In addition to human health impacts caused by ingestion of As-contaminated food, the potential for reduced crop yield due to the accumulation of this toxic metalloid in soil is alarming [2]. Mitigation of As in the food chain has thus received worldwide attention in recent years.

Arsenic accumulation in rice has been recognized as a catastrophe in southeast Asian countries, where rice is the daily staple food [3]. Compared to other cereals, rice is particularly susceptible to As accumulation because it is generally grown under submerged conditions where As mobility is high [4]. Growing rice is mainly dependent on groundwater used for irrigation. In southwest Taiwan, the incidence of elevated concentrations of As in groundwater is known for generating unique cases of endemic Blackfoot disease (BFD), a peripheral vascular disease (i.e., gangrene) [5], which is caused by drinking As-contaminated groundwater. Although the groundwater in the BFD area has not been utilized for drinking since 1990, it is still extensively used for irrigation of croplands, specifically paddies, resulting in the incorporation of this carcinogenic element into the food chain. Research that ensures a safe level of As in rice for consumption is currently very active and includes soil and water management, rhizosphere manipulation, breeding and genetic engineering [3]. Currently, the implementation of As-resistant and/or arsenite [As(III)]-oxidizing bacteria exhibiting PGP traits to lower As toxicity and uptake and support plant growth on As-stressed soils has gained significant attention because compared to other techniques, it is cost-effective and eco-friendly [6].

The fate and behavior of As in paddy soil depends largely on its speciation [4]. In well-aerated soils, arsenate [As(V)] is in a stable form since it is strongly adsorbed on metal (hydr) oxides [7]. Under reducing conditions e.g., in flooded paddy soil, As(V) is reduced to As(III) and becomes the dominant form in the flooded soil [4]. This promotes the incorporation of As(III) into rice since the dominant species taken up by rice roots is As(III) [7]. Oxidation of As(III) leads to the formation of the less toxic and less bioavailable As(V), which has much higher affinity for iron oxides as compared to As(III). This makes As(V) much less mobile than As(III) in soil [7]. It is assumed that the activity of As(III)-oxidizing bacteria may be elevated by root exudates and/or the oxygen released from the rice roots, resulting in more As(V) binding on iron minerals in soil and iron plaque on rice roots. These processes reduce As bioavailability and uptake in rice [1], [3]. In addition, As(III)-oxidation represents a possible means of As detoxification, and As(III)-oxidizing bacteria thus have a vital role in natural As-remediation processes in the environment [8]. Rice grown in As-contaminated soil harbors unique As-resistant micro-flora in the rhizosphere. Such micro-flora may have the advantage of secreting plant growth promoting substances like siderophores, indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and solubilize phosphate to alleviate As-toxicity in rice [9], [10]. The significance of As(III)-oxidizing bacteria exhibiting potential PGP traits can be enormous in As-contaminated paddy soils since these bacteria are able to oxidize the more toxic and more bioavailable As(III) to less toxic and less bioavailable As(V) and are likely to reduce As uptake by rice, increase the tolerance of rice plants against As-stress, stimulate plant growth and contribute in this way to combating a reduction in crop yield in As-contaminated paddy soils.

Although there have been reports on As-resistant/transforming bacteria exhibiting potential PGP traits, the reports on As(III)-oxidizing bacteria exhibiting potential PGP traits and their implementation in soil conditions to evaluate their role in rice plant growth and reduction in As contamination in rice grain remain very elusive (supplementary Table S1). Noteworthy, the implementation of such novel As(III)-oxidizing plant growth promoting bacteria (PGPB) in soil conditions is necessary to get mechanistic interpretation (i.e., the role of this strain on plant growth and As tolerance) and the role of the strain in plant-soil relationships in As-stressed soil [9], [10]. In this study we report (i) isolation and identification of a novel As(III)-oxidizing bacterium from the rhizosphere of rice, (ii) its level of resistance to As(III) and As(V), (iii) detection of aoxB gene and its phylogenetic affirmation, (iv) exhibition of As(III)-oxidase enzyme activity, (v) kinetics of As(III)-oxidation, (vi) quantitative determination of potential PGP traits and (vii) effect of inoculation of As(III)-oxidizing bacteria exhibiting potential PGP traits on plant growth parameters, and As distribution and accumulation in Oryza sativa L.