Active and total rhizosphere microbiomes of plant mixtures diverge from additive expectations, depending on nitrogen and plant composition

Mixing plant functional groups can boost plant productivity, but the underlying rhizosphere microbial dynamics remain unclear. We tested when to expect divergence from additive expectations by measuring plant traits and active and total rhizosphere microbiomes (via BONCAT-FACS and 16S rRNA gene amplicon sequencing) in monocultures and mixtures of legumes, brassicas, and grasses under varying nitrogen. While shoot biomass was predictable from monocultures, legume-containing mixtures yielded greater-than-expected root biomass and N-fixation. Each plant species supported distinct active and total microbiomes, and nitrogen addition increased microbiome divergence among plant species and their mixtures. In most plant mixtures, microbiomes were additive combinations of those associated with the individual species; legume-grass combinations were a notable exception, showing non-additive shifts in microbial composition, microbial activity, and the abundance of Rhizobium sp. This divergence from additive expectations coincided with non-additive biomass gains in legume-grass mixtures, suggesting that emergent plant-microbe interactions may underlie synergistic belowground responses.