Endophytic colonization and nitrogen fixation by Paenibacillus polymyxa in association with lodgepole pine and western redcedar

In this study I provide evidence of biological nitrogen fixation by endophytic, diazotrophic bacteria as a possible source of nitrogen for lodgepole pine (Pinus contorta var. latifolia (Dougl. Engelm.) and western redcedar (Thuja plicata Donn.); conifers that are known for their ability to grow in nitrogen-poor forests of western North America. Diazotrophic bacteria were isolated from root, stem and needle tissues of both tree species, growing on forested sites with contrasting N availability in the interior of British Columbia, Canada. Members of the genera Bacillus and Paenibacillus dominated the culturable, endophytic bacterial community in tissues of both tree species. A Paenibacillus polymyxa isolate strain P2b-2R from lodgepole pine at the nitrogen deficient site near Williams Lake, B.C., demonstrated high (5.1705 μmols C₂H₄/ml), replicable, nitrogenase activity, under laboratory conditions. P. polymyxa strain P2b-2R inoculated and control lodgepole pine and cedar seedlings were grown in a sand – turface mixture enriched with a 5 atom % excess ¹⁵N [Ca(¹⁵NO₃)₂] solution. Root, shoot and seedling length, fresh weight and dry weight demonstrated that both tree species accumulated significantly higher biomass when inoculated with strain P2b-2R. ¹⁵N atom % excess indicated that P2b-2R inoculated lodgepole pine and western redcedar derived 67.53 and 21.94% of their total foliar nitrogen from the atmosphere, respectively. Using in situ confocal laser scanning microscopy, cells of strain P2b-2R tagged with green fluorescent protein were found to colonize the root and stem cortical cells of lodgepole pine, both inter- and intracellularly. Sequences of nif B, H and D genes of strain P2b-2R were obtained using PCR. Phylogenies based on nifH and nifD genes of strain P2b-2R place these genes in monophyletic groups with those of free-living cyanobacteria and root nodule-forming Frankia, respectively. Within the genus Paenibacillus, based on nifH and nifD phylogenies, P. polymyxa was most closely related to P. massiliensis T7, a bacterium isolated from the rhizosphere of willow trees (Salix spp.) in Beijing. These results provide the first evidence of significant endophytic nitrogen fixation in conifer species growing under nitrogen-limited conditions and support the possibility of a novel, ecologically significant interaction between coniferous trees and diazotrophic bacteria.Land and Food Systems, Faculty ofGraduat