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 N-labeled air to trace BNF activities in ΔanfH, ΔnifH, and ΔanfHΔnifH mutants. Assessing bacterial growth, nitrogen content, and N incorporation revealed that BNF is impaired in K. radicincitans DSM16656 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 DSM16656 mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants.

Biological Nitrogen Fixation (BNF) is a process of great importance in crop production systems, as it provides additional natural sources of mineral nitrogen. BNF is catalyzed by diazotrophs that are identified by the nif operon presence comprising the nifH gene that encodes for enzyme nitrogenase synthesis. Thoroughly understanding of factors that influence diazotrophic abundance is crucial for their utilization to enhance sustainability and prevent land degradation in modern agriculture. In this study the impacts of nitrogen fertilization on diazotrophic abundance in Brassica oleracea roots and leaves was investigated in greenhouse experiments by real-time qPCR. One way ANOVA was used to compare means and bivariate Pearson correlation tested for relationships between variables. Increased nitrogen fertilization significantly increased the nitrogen content in leaves but not in roots. No significant changes in nifH gene copy numbers nor in proportion of nifH gene copy numbers were detectable. This indicates no effect of mineral N fertilization on the abundance of total native diazotrophic bacterial numbers in Brassica oleracea plants.