Methylotrophic methanogens and bacteria synergistically demethylate dimethylarsenate in paddy soil and alleviate rice straighthead disease

Microorganisms play a key role in arsenic (As) biogeochemistry, transforming As species between inorganic and organic forms and different oxidation states. Microbial As methylation is enhanced in anoxic paddy soil, producing primarily dimethylarsenic (DMAs), which can cause rice straighthead disease and large yield losses. DMAs can also be demethylated in paddy soil, but the microorganisms driving this process remain unclear. In this study, we showed that the enrichment culture of methylotrophic methanogens from paddy soil demethylated pentavalent DMAs(V) efficiently. DMAs(V) was reduced to DMAs(III) before demethylation. 16S rRNA gene diversity and metagenomic analysis showed that Methanomassiliicoccus dominated in the enrichment culture, with Methanosarcina and Methanoculleus also being present. We isolated Methanomassiliicoccus luminyensis CZDD1 and Methanosarcina mazei CZ1 from the enrichment culture; the former could partially demethylate trivalent DMAs(III) but not DMAs(V) and the latter could demethylate neither. Addition of strain CZDD1 to the enrichment culture greatly accelerated DMAs(V) demethylation. Demethylation of DMAs(V) in the enrichment culture was suppressed by ampicillin, suggesting the involvement of bacteria. We isolated three anaerobic bacterial strains including Clostridium from the enrichment culture, which could produce hydrogen and reduce DMAs(V) to DMAs(III). Furthermore, augmentation of the Methanomassiliicoccus-Clostridium coculture to a paddy soil decreased DMAs accumulation by rice and alleviated straighthead disease. The results reveal a synergistic relationship whereby anaerobic bacteria reduce DMAs(V) to DMAs(III) for demethylation by Methanomassiliicoccus and also produce hydrogen to promote the growth of Methanomassiliicoccus; enhancing their populations in paddy soil can help alleviate rice straighthead disease.

Table S1.Compositions of the methanogenic medium used in the present study.

Figure S1 .
Figure S1.Design of a pair of primers specific for Methanomassiliicoccus based on mtaB gene.A Alignment of mtaB genes obtained from strains or MAGs of Methanomassiliicoccus and the two conserved regions selected for primer design.Agarose gel electrophoresis of mtaB genes amplified with B genome DNA extracted from strain Methanomassiliicoccus luminyensis CZDD1 or luminyensis B10 and Methanosarcina maize CZ1 and C total DNA of paddy soil and methanol enrichment culture as templates to verify the specificity of the primers.D Identification of fragment of mtaB genes via constructing a clone library using the designed primer.

Figure S5 .
Figure S5.Effect of Methanomassiliicoccus luminyensis CZDD1 addition to the methanol enrichment culture on the relative abundance of core genera of methanogens.Data are means ± SD (n = 3).

Figure S6 .Figure S7 .Figure S8 .
Figure S6.Effect of ampicillin addition to the methanol enrichment culture on the relative abundance of bacteria.Only those with an abundance of >0.1% are shown in the Figure.Data are means ± SD (n = 3).

Figure S13 .
Figure S13.Correlations between the percentage of DMAs(V) demethylation and the copy number of mcrA (A, C, E) or mtaB (B, D, F) in all paddy and upland soils (A, B), or within the paddy soils (C, D) or upland soils (E, F).Each symbol represents one replicate of a soil.

Table S2 .
Primers used in the present study.

Table S3 .
Locations and properties of the paddy and upland soils used in the present study.

Table S4 .
Mass balance and distribution of arsenic species in the soil solution and solid phases, and volatile arsenic in the headspace of the methanol enrichment cultures amended with or without DMAs (80 μM x 5 mL = 0.4 μmol per bottle) at the end of the incubation experiment.Data are means ± SE (n = 3). 6