Isolation and identification of diazotrophic bacteria from internal tissues of Pinus contorta and Thuja plicata

Lodgepole pine (Pinus contorta var. latifolia Engelm. ex S. Watson) and western red cedar (Thuja plicata Donn ex D. Don) are capable of growing in soils that are severely nitrogen-limited. A rarely evaluated but possible nitrogen source for gymnosperms growing at nutrient poor sites is nitrogen fixation by endophytic bacteria. To investigate this possibility, we looked for nitrogen-fixing bacteria in surface-sterilized needle, stem, and root tissues of naturally regenerating lodgepole pine and western red cedar seedlings (2-4 years old) and trees (>20 years old) growing near Williams Lake, Chilliwack Lake, and Boston Bar, British Columbia, Canada. Ninety-nine bacterial isolates were obtained by plating surface-sterilized plant tissue extracts on tryptic soy agar (TSA) and combined carbon medium agar (CCMA), a nitrogen-free medium. Bacterial isolates were identified using gas chromatographic fatty acid methyl ester (GC-FAME) and 16S rRNA gene analyses and were evaluated for nitrogenase activity using an acetylene reduction assay. Representatives of the genera Bacillus and Paenibacillus dominated the culturable endophytic bacterial community in tissues of both tree species, with three Paenibacillus isolates and one Dyadobacter fermentans strain demonstrating consistently high acetylene-reduction activities. Our results raise the possibility that endophytic nitrogen fixation provides a supplementary nitrogen source for naturally regenerated lodgepole pine and western red cedar.