Genetic remodeling of soil diazotrophs enables partial replacement of synthetic nitrogen fertilizer with biological nitrogen fixation in maize

Increasing biological nitrogen (N) fixation (BNF) in maize production could reduce the environmental impacts of N fertilizer use, but reactive N in the rhizosphere of maize limits the BNF process. Using non-transgenic methods, we developed gene-edited strains of Klebsiella variicola ( Kv 137-2253) and Kosakonia sacchari ( Ks 6-5687) bacteria optimized for root-associated BNF and ammonium excretion in N-rich conditions. The aim of this research was to elucidate the mechanism of action of these strains. We present evidence from in vitro, in planta and field experiments that confirms that our genetic remodeling strategy derepresses BNF activity in N-rich systems and increases ammonium excretion by orders of magnitude above the respective wildtype strains. BNF is demonstrated in controlled environments by the transfer of labeled 15 N 2 gas from the rhizosphere to the chlorophyll of inoculated maize plants. This was corroborated in several 15 N isotope tracer field experiments where inoculation with the formulated, commercial-grade product derived from the gene-edited strains (PIVOT BIO PROVEN® 40) provided on average 21 kg N ha -1 to the plant by the VT-R1 growth stages. Data from small-plot and on-farm trials suggest that this technology can improve crop N status pre-flowering and has potential to mitigate the risk of yield loss associated with a reduction in synthetic N fertilizer inputs.