Presence and activity of nitrogen-fixing bacteria in Scots pine needles in a boreal forest : a nitrogen-addition experiment

Endophytic nitrogen-fixing bacteria have been detected and isolated from the needles of conifer trees growing in North American boreal forests. Because boreal forests are nutrient-limited, these bacteria could provide an important source of nitrogen for tree species. This study aimed to determine their presence and activity in a Scandinavian boreal forest, using immunodetection of nitrogenase enzyme subunits and acetylene-reduction assays of native Scots pine (Pinus sylvestris L.) needles. The presence and rate of nitrogen fixation by endophytic bacteria were compared between control plots and fertilized plots in a nitrogen-addition experiment. In contrast to the expectation that nitrogen-fixation rates would decline in fertilized plots, as seen, for instance, with nitrogen-fixing bacteria associated with bryophytes, there was no difference in the presence or activity of nitrogen-fixing bacteria between the two treatments. The extrapolated calculated rate of nitrogen fixation relevant for the forest stand was 20 g N ha-1 year-1, which is rather low compared with Scots pine annual nitrogen use but could be important for the nitrogen-poor forest in the long term. In addition, of 13 colonies of potential nitrogen-fixing bacteria isolated from the needles on nitrogen-free media, 10 showed in vitro nitrogen fixation. In summary, 16S rRNA sequencing identified the species as belonging to the genera Bacillus, Variovorax, Novosphingobium, Sphingomonas, Microbacterium and Priestia, which was confirmed by Illumina whole-genome sequencing. Our results confirm the presence of endophytic nitrogen-fixing bacteria in Scots pine needles and suggest that they could be important for the long-term nitrogen budget of the Scandinavian boreal forest