Root colonization and environmental fate of the bioherbicide pseudomonas fluorescens BRG100
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Pseudomonas fluorescens 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 S17-1λ 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. Pseudomonas fluorescens 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). 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. 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. These results suggest that Pseudomonas fluorescens BRG100 has considerable potential as a bioherbicide because of its successful root colonization of green foxtail and wheat. Pseudomonas fluorescens 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 P. fluorescens BRG100.
DegreeMaster of Agriculture (M.Agr.)
DepartmentApplied Microbiology and Food Science
ProgramApplied Microbiology and Food Science
SupervisorKorber, Darren R.; Boyetchko, Susan M.
CommitteeShand, Phyllis J.; Legget, Mary; Hynes, Russell K.; Vujanovic, Vladimir
green fluorescent protein