Characterizing the beneficial ecological association of endophytic bacteria with lodgepole pine trees naturally-regenerating at unreclaimed gravel mining pits

Forest landscapes are commonly affected by human disturbances giving rise to degraded edaphic conditions such as poor soil structure, inadequate plant-nutrients, and in some cases like gravel mining, a complete loss of topsoil. Gravel mining has led to the prevalence of highly-disturbed sites in the central interior region of British Columbia, Canada that have very limited soil nitrogen-levels due to the presence of gravelly-substrates, lack of forest floor, and low atmospheric nitrogen-deposition. Despite such severe conditions, lodgepole pine trees are thriving at these unreclaimed gravel pits, with tissue nitrogen-contents and growth-rates seemingly unaffected by low soil nitrogen-levels, indicating that pine trees could be accessing an unknown nitrogen-source. One possibility could be biological nitrogen-fixation (BNF) by endophytic diazotrophs (nitrogen-fixing bacteria living asymptomatically within plants). Testing this possibility, 77 potential endophytic diazotrophs were isolated from internal tissues of pine trees growing at two gravel pits in this region. Of these, 32 strains showed positive nitrogenase activity in the acetylene reduction assay. Fourteen strains that consistently showed high nitrogenase activity were selected for a year-long greenhouse study to evaluate their ability to sustain pine growth under nitrogen-poor conditions. After one year, all strains colonized the internal tissues of pine and accumulated 23%-53% of the host’s foliar nitrogen via BNF as estimated through a 15N isotope-dilution assay. Furthermore, bacterial inoculation significantly enhanced pine seedling length (31%-64%) and biomass (100%-311%). The presence of the nifH gene was also confirmed in all 14 strains. In addition to BNF, most strains demonstrated considerable potential to promote plant growth via several other mechanisms including phosphate solubilization, siderophore production, ACC deaminase activity, IAA production and lytic enzyme activity. Notably, three Pseudomonas strains (AN1r, AR1r, SN1r) contributed >50% of the host’s foliar nitrogen, showed the highest activity in multiple growth-promoting assays and enhanced seedling biomass by 4-fold. These results suggest that lodgepole pine trees may be dependent on endophytic bacteria for their survival and growth, indicating a unique ecological association that may explain natural pine-regeneration on bare gravel. However, this beneficial association must be validated under field conditions to determine their future value in restoring ecosystem productivity at disturbed sites.Land and Food Systems, Faculty ofGraduat