Non-nodulating Paenirhizobium fix nitrogen in association with cereal roots

Cereal root microbiomes harbour diverse diazotrophic bacteria, yet the taxa capable of sustained nitrogen fixation in association with cereal roots remain poorly characterized. Here, two high-performing nitrogen-fixing strains (B6 and J2) were isolated from barley roots and identified as belonging to the family Rhizobiaceae in the genus Paenirhizobium. Both strains possess plasmid-encoded canonical rhizobial nif and fix genes for nitrogen fixation but lack nodulation genes. Their genomes have a 5.7 Mb chromosome and four repABC plasmids. Unlike most nodulating rhizobia, strains B6 and J2 fixed nitrogen in laboratory culture on a range of carbon sources, achieving maximal activity on organic acids at low ammonium (<0.5 mM) and oxygen concentrations (1-3%). Both strains colonized the total root systems of barley plants, with population densities of 10⁶ CFU g^-1 fresh root weight. Strains fixed high levels of nitrogen on barley plants, similar to or greater than other known free-living diazotrophs. These findings expand the ecological context of rhizobial nitrogen fixation and identify cereal-associated Paenirhizobium as a previously unrecognized component of the diazotrophic cereal root microbiome.