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UBC Theses and Dissertations

Isolation of endophytic bacteria from Lodgepole Pine (Pinus contorta var. latifolia (Dougl.) Engelm.) and Western Red Cedar (Thuja plicata Donn.) and determination of their nitrogen fixing ability Bal, Amandeep S.


The diversity of endophytic bacteria in lodgepole pine and western red cedar tissues and whether endophytic diazotrophs isolated from these tissues were capable of supplying biologically significant amounts of fixed nitrogen to these two species were examined. Plant samples were collected from stands in central and southern British Columbia and endophytic bacteria were isolated primarily from stem tissues using a surface sterilization-trituration-plating technique. Most of the endophytic bacteria isolated from pine and cedar tissues belonged to the genera Bacillus and Paenibacillus. The N-fixing ability of isolates was tested using an acetylene reduction assay. Four isolates capable of reducing acetylene were selected to determine their ability to provide biologically significant amounts of fixed N to pine and cedar seedlings in two separate controlled environment experiments. This included Paenibacillus amylolyticus C3b, rifamycin-resistant derivatives of P. peoriae/polymyxa P2b and P18b, and a rifamycin-resistant derivative of strain P19a, which was closely related to Dyadobacter fermentans. Surface sterilized pine and cedar seeds were sown separately in glass tubes containing an autoclaved mixture of sand and montmorillonite clay treated with a nutrient solution having a ¹⁵N label. Each tube was then inoculated with ca. 10⁷ cfu of one of strains P2b-2R, P18b-2R, P19a-2R, orC3b. The only treatment in which seedlings had significantly lower atom % ¹⁵N excess values in foliage than in control seedlings in both plant growth trials for cedar and pine was the P2b-2R treatment. Inoculation of seed with strain P2b-2R resulted in cedar and pine seedlings harvested 35 and 42 weeks, respectively, after sowing, deriving as much as 56% and 66%, respectively, of their foliar N from the atmosphere. Pine seedlings from the C3b and P18b-2R treatments from only the first trial derived less significant quantities of foliar N from the atmosphere. Strain P19a-2R did not provide significant amounts of fixed N to cedar and pine seedlings in either trial. Despite cedar and pine seedlings from the P2b-2R treatments deriving significant amounts of fixed N, increases in foliar N content were not common and growth of seedlings of both species was depressed. Strain P2b-2R could be readily isolated from rhizosphere samples following both seedling harvests for cedar and pine. However endophytic colonization of tissues by this strain could not be confirmed due to persistent contamination on imprint plates used to determine the effectiveness of the tissue surface sterilization techniques employed.

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