Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica

Leaf surfaces (phyllospheres) have been shown to provide appropriate conditions for colonization by microorganisms including diazotrophic bacteria that are able to fix atmospheric nitrogen (N-2). In this study, we determined leaf-associated N-2 fixation of a range of rainforest plants in Costa Rica, under different environmental conditions, by tracing biomass N incorporation from N-15(2). N-2-fixing bacterial communities of the plant species Carludovica drudei, Grias cauliflora and Costus laevis were investigated in more detail by analysis of the nifH gene and leaf-associated bacteria were identified by 16S rRNA gene analysis. N-2 fixation rates varied among plant species, their growth sites (different microclimatic conditions) and light exposure. Leaf-associated diazotrophic bacterial communities detected on C. drudei and C. laevis were mainly composed of cyanobacteria (Nostoc spp.), whereas on the leaves of G. cauliflora gamma-proteobacteria were dominant in addition to cyanobacteria. The complexity of diazotrophic communities on leaves was not correlated with N-2 fixation activity. 16S rRNA gene sequence analysis suggested the presence of complex microbial communities in association with leaves, however, cyanobacteria showed only low abundance. Our findings suggest that cyanobacteria as well as gamma-proteobacteria associated with leaf-colonizing epiphytes may provide significant nitrogen input into this rainforest ecosystem.