Aboveground N2-fixing bacterial endophytes in subalpine conifers: A novel pathway for ecosystem acquisition of new nitrogen?

Background/Question/Methods

Temperate and boreal coniferous forests accumulate more nitrogen (N) in soil and vegetation than can be explained by known sources of N. While recent research provides compelling evidence for cyanobacterial-moss N₂ fixation and geologic N sources, another potential source of N to these ecosystems is N₂-fixing endophytic bacteria inside non-legumes. Bacterial endophytes, which colonize the interior of plants, are emerging as an important component of the plant microbiome. Their roles in plant growth, nutrient acquisition, and protection against disease and abiotic stress are increasingly being harnessed in agricultural ecosystems, yet, not much is known about how such properties influence plants in the wild. We asked whether the Pinus flexilis (limber pine) and Picea engelmanni (Engelmann spruce) growing in the subalpine environment at Niwot Ridge, CO, host a consistent microbiota of potentially N₂-fixing bacteria in their needles. Using 16S rRNA pyrosequencing, we characterized the endophytic needle communities in multiple individuals of each species at two elevations; at treeline, and near the local warm edge limit of the subalpine forest distribution.

Results/Conclusions

In contrast to previous studies on the endophytic community of Arabidopsis and Poplar, we found remarkably low variability in the endophyte species composition among and within individuals, and between conifer species. Both conifers hosted the same ‘core’ of bacterial species within their needles, with a few phylotypes in the family Acetobacteriacace dominating both species. The most prominent phylotype in all samples of both hosts (36 and 21 % of the total sequences from P. flexilis and P. engelmani, respectively) is similar to Gluconacetobacter diaztrophicus, an N₂-fixing endophyte in sugarcane, and may represent a novel and locally adapted N₂-fixing symbiosis in these conifers. There may be a correlation between N fertility and relative abundance of N₂-fixers, because two samples from treeline  (where low temperatures slow conversion of organic to inorganic forms of soil N) showed a higher relative abundance of dominant potential N₂-fixers. Using acetylene reduction, we demonstrated nitrogenase activity in twigs from P. flexilis growing at treeline. The presence of N₂-fixing symbioses in conifers could help explain how conifers can grow in nutrient-limited soil, and why boreal and temperate ecosystems have been found to accumulate more N than can be accounted for by known N input pathways.