Endophytic nitrogen fixation – a possible ‘hidden’ source of nitrogen for lodgepole pine trees growing at unreclaimed gravel mining sites

Lodgepole pine (Pinus contorta var. latifolia) trees have been thriving on unreclaimed gravel mining sites in British Columbia, Canada, with tissue nitrogen-content and growth-rate unaffected by extremely low soil nitrogen-levels. This indicates that pine trees could be accessing a hidden nitrogen source to fulfill their nitrogen requirements - possibly via endophytic nitrogen-fixation. Endophytic bacteria originally isolated from native pine trees growing at gravel sites were selected (n = 14) for in vitro nitrogen-fixation assays and a year long greenhouse study to test the overall hypothesis that naturally occurring endophytic nitrogen-fixing bacteria sustain pine tree growth under nitrogen-limited conditions. Each of the 14 bacteria colonized the internal tissues of pine trees in the greenhouse study and fixed significant amounts of nitrogen from atmosphere (23%-53%) after one year as estimated through 15N isotope dilution assay. Bacterial inoculation also significantly enhanced the length (31%-64%) and biomass (100%-311%) of pine seedlings as compared to the non-inoculated control treatment. In addition, presence of the nifH gene was confirmed in all 14 bacteria. Our results support the possibility that pine trees associate with nitrogen-fixing bacteria, capable of endophytic colonization, to survive at unreclaimed gravel mining pits and this association could potentially be utilized for effective reclamation of highly disturbed sites in a sustainable manner.