<i>Pseudomonas fluorescens</i> BRG100 produces secondary metabolites with herbicidal activity to the grass weeds wild oat, Avena fatua, and green foxtail, Setaria viridis. The green fluorescence protein (gfp) gene was introduced into P. fluorescens BRG100 from Escherichia coli S17-1¥ë via a Tn5 mini transposon suicide vector system. Colony morphology, growth rate in liquid media, weed biocontrol efficacy (plant growth pouch), carbon utilization (Biolog GN) and root colonization of green foxtail by several P. fluorescens BRG100gfp transformants were determined to be the same as the wild type. <i>Pseudomonas fluorescens</i> BRGgfp-15 was found to be most similar to the wild-type in all of the above characteristics and was thus used in subsequent experiments. Note: all strains of Pseudomonas fluorescens will be referred to by only their strain throughout (ie. BRGgfp-15 and BRG100). <p>It was determined by population dynamics per section of root with spiral plating on culture medium, epi-fluorescence and confocal microscopy that BRGgfp-15 colonized all areas of the root, but showed a preference for the proximal 1/3 section and the seed. In the proximal section the mean number of viable cells per gram dry weight was log109.06 and log109.31, when applied as liquid inoculum and as the pesta granular formulation, respectively. With liquid inoculum there was only log107.53 viable cells/g in the middle 1/3 section and log107.01 viable cells/g in the distal 1/3 section. The number of viable cells/g with pesta granules was log107.61 and log107.34, for the middle and distal sections, respectively. The root hairs, root tip, and ventral portion of the seed were all areas of heavy colonization relative to the other areas of the root. <p>Survival of BRGgfp-15 in the pesta formulation was examined in 2 soil types, clay and clay loam, in a thermogradient plate apparatus by a factorial randomized design complete block experiment. The experiment included: 3-12 hour diurnal temperature regimes: 5-15¨¬C, 15-25¨¬C, and 25-35¨¬C and 3 moisture levels: 25, 50 and 75% of soil moisture holding capacity. Sampling was carried out after 0, 14, 28 and 42 days. The highest numbers of viable BRGgfp-15 cells/g were found in the pesta granules in soil subjected to the lowest diurnal temperature regime and moisture content. The lowest numbers of viable cells/g were found in the pesta granules incubated in the highest diurnal temperature and moisture. This suggests lower soil temperature and moisture enhances survival of BRGgfp-15 in pesta and/ or higher soil temperature and moisture enhances the release and dissemination of BRGgfp-15 from pesta granules. When subjected to a 5-15¨¬C-temperature regime the number of viable cells/g was log109.80. When subjected to 15-25¨¬C the viable cells/g was log108.96 and with 25-35¨¬C it was log107.33. The mean number of viable cells/g was log109.36, log108.86, and log107.87, for 25, 50, and 75% soil moisture holding capacity, respectively. There was also a significantly higher number of viable cells/g in the clay soil collected from Saskatoon, log109.00, as compared to the clay loam soil collected from Scott, which was log108.40. <p>These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. <i>Pseudomonas fluorescens</i> BRGgfp-15 survived well under various environmental conditions when formulated into pesta granules, proving the pesta formulation was an excellent formulation. In addition, gfp was shown to be an excellent conservative marker for monitoring the root colonization and survival of <i>P. fluorescens</i> BRG100.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:SSU.etd-09172008-190701 |
| Date | 22 September 2008 |
| Creators | Hanson, Caressa |
| Contributors | Shand, Phyllis J., Legget, Mary, Korber, Darren R., Hynes, Russell K., Boyetchko, Susan M., Vujanovic, Vladimir |
| Publisher | University of Saskatchewan |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-09172008-190701/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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