Non-symbiotic nitrogen-fixing bacterial diazotrophs closely associated with the roots of grasses probably contribute most of the new nitrogen acquired to sustain productive natural grasslands, yet their ecology is poorly understood, especially in southern Africa. We looked for genetic evidence, using qPCR and gel electrophoresis, for the presence of the bacterial nifH gene associated with the roots of four grass species (20 plants each) in a mesic grassland in South Arica, which would indicate the potential for N fixation by diazotrophs. Grasses most tolerant of low N (Aristidajunciformis) were predicted to harbour the most diazotrophs, especially compared to those most responsive to fertiliser N (Eragrostiscurvula). However, the nifH gene was found in all 80 root samples and did not differ in copy number between species. Sequencing of a representative sample confirmed the identity of the nifH gene. The recently burned half of the grassland had 60% more nifH genes than the area burned 15 months previously, suggesting that grass growth stimulated by fire could recruit diazotrophs. Given their ubiquity and importance in the N economy of grasslands, research is required to characterise root-associated diazotroph communities, quantify their N fixation rates, and understand their environmental controls.

1: Soil chemical characteristics of composite samples (n = 10 1 ) of topsoil (150 mm depth) from the 'Old' and 'New' positions of the study site. Soil analyses were undertaken by the Analytical Services Sub-directorate of the KwaZulu-Natal Department of Agriculture and Rural Development, Cedara, Hilton, South Africa (Manson et al. 2020).1 Soil samples comprised a composite of the soil shaken off the roots from a random selection of 10 of the tufts examined for diazotrophs in the Old' and the 'New' positions of the study site.

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