Isolation and identification of endophytic diazotrophs from lodgepole pine trees growing at unreclaimed gravel mining pits in central interior British Columbia, Canada

Unreclaimed gravel mining pits located in the central interior of British Columbia have very limited soil N-levels due to gravelly-textured soils with weak profile development, no organic forest floor, and low atmospheric nitrogen (N) inputs through precipitation. However, lodgepole pine (Pinus contorta var. latifolia) trees can be found growing well at these pits with tissue N-content and growth rate unaffected by extremely low soil N-levels, indicating that pine trees are able to meet their N-requirements from an unknown source. We hypothesized that biological N-fixation by bacteria living in the tree tissues (known as endophytic diazotrophs) could be a potential N source for these trees. To test this hypothesis, we isolated 77 potential endophytic diazotrophs from needle, stem and root tissues of pine trees on N-free culture media. Of these, 32 bacteria showed positive N-fixing ability when tested for nitrogenase enzyme activity using the acetylene reduction assay. These endophytic N-fixing bacteria were identified as mainly belonging to the genera Pseudomonas, Bacillus, Paenibacillus, and Rhizobium, which are well-known for their plant-beneficial traits including N-fixation. Therefore, it can be concluded that pine trees growing at these severely N-limited gravel pits naturally harbour endophytic diazotrophs, which could be involved in sustaining their vigorous growth, possibly through biological nitrogen fixation.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author