Abstract
Most attempts to monitor the effects of introductions of Genetically Modified Micro-organisms (GMMs) have centred on the enumeration of specific populations. However for a significant perturbation to be measured, changes of between 100% and 300% (0.3 and 0.5 on a log scale) are necessary for the impact to be significant. Standard population measurements, assessing the survival, dissemination and effect on total indigenous populations do not give an indication of the functioning of the ecosystem. A range of soil enzyme assays have been developed as alternatives to population measurements. Assays for determining chitobiosidase, N-acetyl glucosaminidase, beta-glucosidase, beta-galactosidase, acid phosphatase, alkaline phosphatase, phosphodiesterase, aryl sulphatase and urease activities from small soil samples were developed. These assays were employed to assess the impact of microbial inoculation into the rhizosphere of crop plants and compared to traditional microbial population measurements. The impact of a chromosomally modified Pseudomonas fluorescens (SBW25) in the wheat rhizosphere using a large intact core microcosm was studied with a combined substrate addition of urea, colloidal chitin and glycerophosphate. The substrate addition caused an increase in the soil chitobiosidase, N-acetyl glucosaminidase aryl sulphatase and urease activities but did not affect acid and alkaline phosphatase and phosphodiesterase activity. Seed inoculated with P. fluorescens caused significant increases in rhizosphere chitobiosidase and urease activities and a significant decrease in alkaline phosphatase activity. Inoculation with the bacteria in the presence of substrate gave opposing effects to those treatments without substrate addition. Using these enzyme assays perturbations of less than 20% could be detected. Two strains of Pseudomonas fluorescens were compared in microcosm experiments one with a functional modification of strain F113 with repressed production of the antibiotic 2,4-diacetylphloroglucinol (DAPG), to create the DAPG negative strain F113 G22. The other, SBW25 EeZY 6KX, with nonfunctional modifications consisting of marker genes (LacZY, xylE and kan') only. Both were assessed, along with the corresponding wild types (F113 and SBW25), for their effects upon the indigenous microflora, plant growth and rhizosphere soil enzyme activities. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists). The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations. The survival of the F113 strains were an order of magnitude lower than the SBW25 strains. The DAPG+ strain caused a significant decrease in the shoot to root ratio in comparison to the control and other inoculants. The F113 (DAPG+) inocula resulted in higher alkaline phosphatase, phosphodiesterase and aryl sulphatase activities than the other inoculants and lower beta glucosidase, beta galactosidase and N-acetyl glucosaminidase activities. These results indicate that the soil enzymes are extremely sensitive to perturbations in the rhizosphere ecosystem and are sensitive enough to measure the impact of GMM inoculation. (Abstract shortened by ProQuest.).