Mature temperate and boreal forest are nitrogen (N) limited, yet N budgets indicate unknown sources of N in these ecosystems. Symbiotic N2-fixing plants are notably absent from coniferous forests, and sources to overcome N limitation are not well understood, but include epiphytic N2-fixation in mosses, free-living N2, fixation in litter and soil, bedrock nitrogen, and deposition of nitrogen pollution.
Via Illumina sequencing of the 16S RNA gene, we have found a consistent association between pines growing in nutrient limited ecosystems and specific bacteria, most notably potential N2-fixing acetic acid bacteria (AAB), and Rhizobiales spp. These associations appear to be conserved across host species, time, and geographic distance, suggesting selection on the part of the tree, the bacteria, or both, potentially reflecting a functional partnership based on N2-fixation. Using the acetylene reduction assay on surface sterilized foliar samples, we have confirmed nitrogenase activity in the subalpine conifer Pinus flexilis (limber pine) growing at Niwot Ridge, Colorado, as well as more recently, in in bishop pine (Pinus muricata) and lodgepole pine (Pinus contorta ssp. bolanderi and ssp. contorta) growing along a gradient in soil age and associated variation in soil fertility at the “ecological staircase” in Mendocino, California.
Not surprisingly, N2-fixation rates of needle endophytes are much lower than those of nodulating N2-fixers, and comparable to rates of free-living fixation in soils in temperate and boreal ecosystems. So far, we have found no evidence that local differences in soil N availability affect rates of foliar N fixation. Together, these results suggest that foliar endophytes represent a low-cost, evolutionarily stable N2-fixing strategy for long-lived conifers that never fully alleviates N limitation in temperate and boreal ecosystems. These results open up the possibility that hidden symbiotic N2-fixers hide in other N-poor ecosystems as well.