Methanol utilizers of the rhizosphere and phyllosphere of a common grass and forb host species.

BACKGROUND: Managed grasslands are global sources of atmospheric methanol, which is one of the most abundant volatile organic compounds in the atmosphere and promotes oxidative capacity for tropospheric and stratospheric ozone depletion. The phyllosphere is a favoured habitat of plant-colonizing methanol-utilizing bacteria. These bacteria also occur in the rhizosphere, but their relevance for methanol consumption and ecosystem fluxes is unclear. Methanol utilizers of the plant-associated microbiota are key for the mitigation of methanol emission through consumption. However, information about grassland plant microbiota members, their biodiversity and metabolic traits, and thus key actors in the global methanol budget is largely lacking. RESULTS: We investigated the methanol utilization and consumption potentials of two common plant species (Festuca arundinacea and Taraxacum officinale) in a temperate grassland. The selected grassland exhibited methanol formation. The detection of 13C derived from 13C-methanol in 16S rRNA of the plant microbiota by stable isotope probing (SIP) revealed distinct methanol utilizer communities in the phyllosphere, roots and rhizosphere but not between plant host species. The phyllosphere was colonized by members of Gamma- and Betaproteobacteria. In the rhizosphere, 13C-labelled Bacteria were affiliated with Deltaproteobacteria, Gemmatimonadates, and Verrucomicrobiae. Less-abundant 13C-labelled Bacteria were affiliated with well-known methylotrophs of Alpha-, Gamma-, and Betaproteobacteria. Additional metagenome analyses of both plants were consistent with the SIP results and revealed Bacteria with methanol dehydrogenases (e.g., MxaF1 and XoxF1-5) of known but also unusual genera (i.e., Methylomirabilis, Methylooceanibacter, Gemmatimonas, Verminephrobacter). 14C-methanol tracing of alive plant material revealed divergent potential methanol consumption rates in both plant species but similarly high rates in the rhizosphere and phyllosphere. CONCLUSIONS: Our study revealed the rhizosphere as an overlooked hotspot for methanol consumption in temperate grasslands. We further identified unusual new but potentially relevant methanol utilizers besides well-known methylotrophs in the phyllosphere and rhizosphere. We did not observe a plant host-specific methanol utilizer community. Our results suggest that our approach using quantitative SIP and metagenomics may be useful in future field studies to link gross methanol consumption rates with the rhizosphere and phyllosphere microbiome.

Site-directed mutagenesis to deactivate two nitrogenase isozymes of Kosakonia radicincitans DSM16656T.

Biological nitrogen fixation (BNF) is considered one of the key plant-growth-promoting (PGP) factors for diazotrophic organisms. Whether the iron and iron-molybdenum nitrogenases of Kosakonia radicincitans contribute to its PGP effect is yet to be proven. Hence, for the first time, we conducted site-directed mutagenesis in K. radicincitans to knock out anfH and (or) nifH as a mean to deactivate BNF in this strain. We used 15N2-labeled air to trace BNF activities in ΔanfH, ΔnifH, and ΔanfHΔnifH mutants. Assessing bacterial growth, nitrogen content, and 15N incorporation revealed that BNF is impaired in K. radicincitans DSM16656T ΔnifH and ΔanfHΔnifH. However, we detected no significant contribution of the Fe nitrogenase to biological dinitrogen assimilation under our pure bacterial culture experimental conditions. Such nondiazotrophic K. radicincitans DSM16656T mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants.

Enterobacter radicincitans sp. nov., a plant growth promoting species of the family Enterobacteriaceae.

A plant growth promoting bacterial isolate (D5/23T) from the phyllosphere of winter wheat, able to fix atmospheric nitrogen and to produce auxines and cytokinins was investigated in a polyphasic taxonomy approach. Phylogenetic analyses using the 16S rRNA gene sequence of the strain clearly indicated that the strain belonged to the family Enterobacteriaceae, most closely related to Enterobacter cloacae with 99.0% and Enterobacter dissolvens with 98.5% sequence similarity. Phylogenetic analysis derived from the sequence of the rpoB gene showed the highest sequence similarities to Enterobacter cowanii (93.0%) but supported the distinct position of strain D5/23T. The isolate produced a fatty acid pattern typical for members of the family Enterobacteriaceae. On the basis of the phylogenetic analyses, DNA-DNA hybridizations, and the unique physiological and biochemical characteristics, we propose that strain D5/23T represents a new species of the genus Enterobacter for which we propose the name Enterobacter radicincitans sp. nov.