Christopher Chanway

Paenibacillus polymyxa P2b-2R is a bacterium that originated from the internal stem tissues of lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedlings growing in the interior of British Columbia. Several studies have suggested that P. polymyxa P2b-2R can fix nitrogen in association with lodgepole pine and can promote plant growth. To further evaluate this strain, P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, was generated and used in a long term inoculation study. To confirm its ability to fix N₂ and to contribute to the N nutrition of pine, as well as comparing the similarities or differences between GFP-labeled and wild-type P2b-2R, a one-year study on bacterial colonization and lodgepole pine seedling growth responses to inoculation with wild-type or GFP-labeled P. polymyxa P2b-2R, was performed. Surface-sterilized lodgepole pine seeds were sown in glass tubes containing an autoclaved sand/montmorillonite clay mixture that contained a nutrient solution with a small amount of nitrogen labeled with ¹⁵N as Ca(¹⁵NO₃)₂ (5 % ¹⁵N label). After sowing, seeds were inoculated with either P. polymyxa P2b-2R or P. polymyxa P2b-2Rgfp. Non-inoculated controls received phosphate-buffered saline. The results indicated that both P2b-2R and P2b-2Rgfp were: (i) able to form persistent rhizospheric and endophytic populations, (ii) capable of in situ N fixation and (iii) enhancing seedling growth continuously after four months. Seedlings inoculated with P2b-2R derived 13%-40 % of foliar N from the atmosphere, while those treated with P2b-2Rgfp derived 18%-47 % of foliar N from the atmosphere during seedling growth period. There was a temporary seedling growth reduction in P2b-2R treatments two months after sowing, but seedlings recovered from the growth reduction thereafter. However, P2b-2Rgfp treatments did not display such a decline. Seedlings treated with P2b-2Rgfp had generally higher endophytic populations, foliar N concentrations, %N derived from atmosphere and dry weights, but lower rhizospheric populations than those treated with P2b-2R. However, these differences were not significant. GFP did not affect N-fixation and growth promotion of wild-type P2b-2R in general, however, GFP-labeled P2b-2R might have short-term advantages in early pine growth stages but these are not significant in the longer term.

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Paenbacillus polymyxa strain P2b-2R has previously been shown to colonize lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) internal tissues and stimulate seedling growth possibly through N₂ fixation. I evaluated the biochemical characteristics of P. polymyxa strain P2b-2R and the effects of soil nitrogen (N) concentrations on lodgepole pine seedling growth after inoculation with P2b-2R. The bacterium was capable of using major plant cell wall components including carboxymethyl cellulose, xylan and sodium polypectate in addition to 39 of 95 carbon sources from BioLog GP2 microplates.These results suggest that hydrolytic enzymes may facilitate endophytic colonization by P2b-2R. Bacterial colonization and lodgepole pine seedling growth responses (foliar ¹⁵N atom % excess, foliar N concentrations, total foliar N and root and shoot biomass) to inoculation with P2b-2R were assessed at varying soil N concentrations in a one-year greenhouse study. Surface sterilized pine seeds were sown in Ray Leach cone-tainers containing an autoclaved sand-montmorillonite clay mixture and inoculated with log 6 colony forming units (cfu) P2b-2R. Non-inoculated controls received phosphate-buffered saline. Seedlings were then subjected to monthly applications of a nutrient solution containing one of four soil N concentrations as Ca(NO₃)₂ (5% ¹⁵N label): 0.0029, 0.029, 0.29 and 2.9 mmol L-¹, referred to as ‘very low’, ‘low’, ‘medium’ and ‘high’ soil N treatments, respectively. Rhizospheric and endophytic population sizes of P2b-2R, foliar ¹⁵N atom % excess and foliar N concentrations were not significantly affected by P2b-2R during the experiment. Inoculation with P2b-2R resulted in seedling biomass across the four soil N treatments four months after sowing. However, seedling growth inhibition was transient as it was not detectable four months later and by month twelve, inoculated seedlings from the very low soil N treatment had accumulated 56.3 and 46.4% more root and shoot biomass, respectively, than controls. Seedlings from the medium soil N treatment responded similarly to bacterial inoculation, but no biomass enhancement was observed at low and high soil N concentrations. My results suggest that pine biomass stimulation by P2b-2R may depend on soil N concentrations and that P2b-2R can enhance biomass accumulation of pine seedlings without providing significant amounts of fixed N.

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Green fluorescent protein (GFP) as a marker gene has facilitated biological research in plant-microbe interactions. However, our knowledge about the effects of introduction of this marker protein in a microbe is very limited. I analyzed the effect of GFP-tagging of a plant growth-promoting bacterium, Paenibacillus polymyxa P2b-2R, on its ability to fix nitrogen and promote growth of important crop species: corn, canola and tomato. To evaluate this, corn, canola and tomato seeds were inoculated with P2b-2R and P2b-2Rgfp and grown for 40 days. Seedlings were harvested 20, 30 and 40 days after inoculation for evaluation of rhizospheric and endophytic colonization (cfu). Biological nitrogen fixation (¹⁵N dilution) and growth response (length and biomass) of P2b-2Rgfp inoculated seedlings was also compared with control and P2b-2R inoculated seedlings. The entire experiment was repeated to confirm the treatment effects. P2b-2Rgfp inoculated seedlings (i) accumulated upto 55% more biomass than non-inoculated controls and 17% more biomass than P2b-2R treated seedlings; (ii) were upto 41% longer than controls and 17% longer than P2b-2R treated seedlings; and (iii) fixed 18% of nitrogen from atmosphere (5% higher than P2b-2R). Canola seedlings inoculated with P2b-2Rgfp (i) had 118% more biomass than controls and 69% more biomass than P2b-2R treated seedlings; (ii) were 69% longer than controls and 37% longer than P2b-2R treated seedlings; and (iii) derived 22% of nitrogen from atmosphere (6% more than P2b-2R). But in case of tomato, P2b-2Rgfp and P2b-2R inoculated seedlings were analogous in terms of growth promotion and nitrogen fixation. Seedlings inoculated with either P2b-2Rgfp or P2b-2R were nearly 40% longer than controls, assimilated nearly 90% more biomass than controls, and fixed nearly 17% of nitrogen from atmosphere. P2b-2Rgfp and P2b-2R strains formed consistent rhizospheric and endophytic populations in corn, canola and tomato roots. To the best of my knowledge, these results represent the first evidence that transformation of a bacterial strain by GFP can significantly enhance its plant growth-promoting efficacy in corn and canola. Since the results of tomato were contrary to corn and canola, it can also be concluded that effect of GFP-tagging of P2b-2R might vary depending on the host plant.

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Availability of nitrogen is the most yield-limiting mineral factor in crop production. Several Paenibacillus bacterial strains that were able to fix nitrogen from atmosphere were isolated from extracts of surface-sterilized lodgepole pine seedling and tree tissues. One strain, Paenibacillus polymyxa P2b-2R, was found to derive high amounts of nitrogen from the atmosphere when introduced into gymnosperm species, namely lodgepole pine and western red cedar. I wanted to determine if Paenibacillus polymyxa P2b-2R could colonize, fix nitrogen and promote the growth of important agricultural crops such as corn and canola. For this I inoculated corn and canola seeds with P. polymyxa P2b-2R and grew them for 30 and 60 days, respectively. Corn seedlings were harvested 10, 20 and 30 days after inoculation and canola seedlings were harvested 20, 40 and 60 days after inoculation for evaluation of rhizospheric and internal tissue (endophytic) colonization (cfu). Seedlings were also evaluated for biological nitrogen fixation (¹⁵N dilution) and growth promotion (length and biomass) at these harvest intervals. The entire experiment was repeated to confirm the treatment effects. P2b-2R successfully colonized the corn rhizosphere with 5.52 x 10⁶ cfu/g dry root and the canola rhizosphere with 1.08 x 10⁸ cfu/g dry root. P2b-2R also colonized internal tissues of corn and canola root with population densities of 4.54 x 10⁵ cfu/g fresh weight and 2.36 x 10³ cfu/g fresh weight, respectively. Corn seedling growth was promoted significantly by inoculation with P2b-2R with an increase of 35% in length and 31% in biomass, 30 days after inoculation. Similarly, canola seedling length was promoted by 25% and biomass by 30%, 60 days after inoculation. Corn seedlings inoculated with P2b-2R were able to derive about 17% of foliar nitrogen from the atmosphere, 30 days after inoculation. Similarly canola seedlings derived nearly 20% of foliar nitrogen from atmosphere, 60 days after inoculation. These results clearly suggest that Paenibacillus polymyxa P2b-2R might have a broad range host capability and is able to fix nitrogen and promote the growth of at least certain important agricultural crops – corn and canola.

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