Nitrogen fixation in pine: Direct confirmation and location of the diazotrophic population

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Nitrogen is often the most limiting plant nutrient in natural ecosytems rendering a distinct advantage to species able to provide their own supply through biological nitrogen fixation (BNF). Plant-associated BNF can satisfy part of the plant N requirement in certain non-nodulating herbaceous species but the only known non-nodulating plants that can obtain most plant N from BNF (up to 80%) comprise a few cultivars of sugarcane.The causative agent of BNF in sugarcane was initially thought to be an endophytic strain of Gluconoacetobacter diazotrophicus, but more recent studies indicate that the primary source of diazotrophy may involve a consortium of bacteria, possibly including uncultured strains, that live on or inside plant tissues. Based on reports than the microorganism responsible for BNF in sugarcane and other herbaceous plant species were culturable endophytes, I searched for similar microorganisms in the commercially important gymnosperm, lodgepole pine and isolated several culturable Paenibacillus polymyxa strains that possessed significant acetylene reduction activity from extracts of surface-sterilized lodgepole pine seedling and tree tissues. Pine seedlngs treated with one of these strains, P2b-2R, derived up to 79% of foliar N from BNF and accumulated significantly more biomass than controls. My results so far suggest that (i) pine seedlings can derive almost all foliar N from nitrogen fixation by P2b-2R, (ii) BNF by P2b-2R causes seedling growth promotion, if seedlings are grown for a sufficiently long time and (iii) P2b-2R's endophytic habit facilitates BNF. However, the experiments conducted so far were exploratory in nature and did not measure N2 fixation directly or rule out other possible causes of seedling growth promotion. I propose to confirm our indirect evidence of BNF by P2b-2R in pine using a 15N tissue incorporation assay and to determine if pine seedling growth promotion by this microorganism results from BNF using a P2b-2R nif- derivative of the wild type. In addition, because P2b-2R colonizes internal and external root, stem and needle tissues, I propose to determine which of these plant-associated N2 fixing populations contributes most to pine-associated BNF.