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Tuberculate ectomycorrhizae on Lodgepole Pine (Pinus contorts) and associated nitrogen fixation Paul, Leslie Robin


Nitrogenase activity associated with tuberculate ectomycorrhiza (TEM) on Pinus contorta in the central interior of British Columbia was investigated. In situ measurements of nitrogenase activity were conducted using a modification of the acetylene reduction assay (ARA). Nitrogenase activity, as measured by ethylene (C₂H₄) production from T EM revealed that average amounts of C₂H₄ produced per gram of Suillus tomentosus TEM for a twenty-four hour period ranged from 0.00 nmoles to 5696.77 nmoles from all sites over a two year period. The maximum amount of C₂H₄ produced per gram of S. tomentosus TEM for a twenty-four hour period was 25098.91 nmoles. Average nmoles C₂H₄ g⁻¹ TEM 24h⁻¹ differed significantly between class 2 stands (young, < 40 years old) and class 8 stands (old, >140 years old)(p=0.004). Estimated nitrogen contribution from S. tomentosus TEM could be significant to the nitrogen budget of P. contorta stands within the SBPSxc biogeoclimatic subzone of British Columbia. Using DNA sequence analysis and morphological analysis, the fungal symbiont of TEM on P. contorta was identified as the species S. tomentosus. There were two species of N₂-fixing bacteria isolated from interstitial tissue of S. tomentosus TEM. Use of gas chromatographic fatty acid methyl ester analysis and 16S rDNA analysis revealed that the two species were Paenibacillus amyloyticus and Methylobacterium mesophillicum. Both species tested positive for nitrogenase activity in vitro and both species contained a nifH amplicon. S. tomentosus TEM contain a haemoprotein with a similar isoelectric point as haemoglobin that may acting in a similar physiological manner as leghaemoglobin in root nodules. This is the first report of a haemoglobin associated with a fungus. Complete formation of S. tomentosus TEM appears to be influenced by some factor from the N2-fixing bacteria found within the interstitial tissue. S. tomentosus TEM symbiosis is a tripartite relationship which involves the N₂-fixing bacteria, mycorrhizal fungus and host tree. This tripartite relationship appears to be functionally and morphologically similar to root nodules on legume and non-legume plants. Nitrogen fixation associated with S. tomentosus TEM is possibly a significant input to the nitrogen budgets of P. contorta.

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