Effects of herbivores on nitrogen fixation by grass endophytes, legume symbionts and free-living soil surface bacteria in the Serengeti

Grass roots can harbor abundant endophytic N₂-fixing microbes (diazotrophs), but their abundance and activity compared to those on legumes and in soil crusts is still unknown. Here, in a natural ecosystem, the Serengeti of East Africa, we explored whether herbivores and soil nutrients limited grass root endophyte diazotroph abundance and their root mass-specific and area-specific N₂-fixation, as they often do for diazotrophs symbiotic with legumes and those free-living in soil. N₂-fixation and copy number of the nitrogenase gene nifH was measured with stable isotope and molecular methods, respectively, for the dominant grass Themeda triandra, and legume, Indigofera volkensii, and in the top 5 cm of soil in a 16-year herbivore exclosure experiment across four sites that varied in mean annual rainfall and soil N, P, and moisture. T. triandra nifH gene copy number was highly variable across sites and individuals but often approached or exceeded that of I. volkensii roots and soils. T. triandra roots generally exhibited lower root mass-specific N₂-fixation (activity), which was not reduced by herbivores and increased in drier soils. In contrast, I. volkensii activity was only reduced by herbivores and soil diazotrophs were mostly inactive. T. triandra exhibited greater area-specific N₂-fixation than I. volkensii, due to its much greater root biomass, but this difference was reduced by herbivores. Grass-associated endophytic diazotrophs may fix far more N₂ in natural systems than previously realized, and may be limited by different factors those affecting symbiotic legume and free-living soil diazotrophs.