High-latitude and-altitude ecosystems are typically nitrogen (N)-limited due to low rates of N input and rapid microbial immobilization of inorganic N, and temperate and boreal forest trees must use a combination of sources and pathways to meet their N demand, including uptake of multiple forms of N, and N transfer via ectomycorrhizal fungi. Recent findings suggest that both evergreen and deciduous trees have the potential to directly access atmospheric N via N2-fixing bacteria in foliage and roots, and that the bacteria responsible may be conserved across sites and conifer species. Here, to test if these patterns are generalizable across conifer species (in the genera Pinus, Picea, and Abies) and forest sites, and to identify the factors that influence variation in N2-fixation rates, we investigated rates of N2-fixiation and characterized foliar microbiomes in wider set of conifer species in subalpine and coastal N limited sites. We used a combination of Illumina sequencing of the 16S RNA gene and single cell genome sequencing to characterize the foliar surface and endosphere bacterial communities. We used the acetylene reduction to test for nitrogenase activity in branch tips of mature trees, and 15N labeling to test for enrichment of fixed N in whole saplings.
Results/Conclusions
Our 16S rRNA and single cell genome data reveal both consistency in the community, and variation across sites. Acetic acid bacteria dominated needle surface across sites, while gammaproteobacteria (especially in the family Pseudomonadacae) dominated the endosphere community in some sites. Single cell genomes match the taxa identified by 16S rRNA sequencing, but genome recovery was incomplete, and we have yet to identify bacteria responsible for N2-fixation; however, the partial genomes show signatures of plant association, encoding e.g. secretion systems and eukaryote-like domains. N2-fixation rates in Pinus contorta and Pinus muricata varied significantly with soil N availability across a marine terrace chronosequence in CA. Rates also varied significantly between species and were significantly higher in 2017, a high precipitation year, than in 2016, a moderate drought year. Our preliminary data from a 15N2 labeling experiment on saplings showed enrichment of 15N in both foliage and roots, suggesting that tree-associated N2-fixation occurs both above- and belowground. Taken together, our results suggest an association that involves selection on the part of the tree, the bacteria, or both, reflecting a functional partnership based on N2-fixation, with variable rates of N2-fixation varies across tree species and environments.