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Title: Soil enzymes as indicators of perturbations in the rhizosphere.
Author: Naseby, David Craig.
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1996
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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, ß-glucosidase, ß-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 andglycerophosphate. 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 Pseudonionas 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 rhizospheres oil enzymea ctivities.S ignificantp erturbationsw ere found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategistsThe 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 ß glucosidase, ß 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. The impact of the biocontrol agent Pseudomonas fluorescens F113 upon soil biochemistry was evaluated in sugar beet field trials in conjunction with UC Cork and Irish Sugar. The inoculum established as an effective population size greater than 5x105 bacteria per root system upon the harvest of the sugar beet, 3 months after sowing. Phosphorus cycle enzymes (acid phosphatasea, lkaline phosphatasaen d phosphodiesterasea)l,o ng with aryl sulphatase activity were negatively correlated with the soil phosphate content. Urease activity was positively correlated with the phosphate content. No significant differences were found that could be attributed to the experimental treatments. This indicates that either the effect of the treatmentwas small, or the variation in field chemistry was greater than perturbations that may have been caused by the field trial treatments. The Pseudomonasfl uorescens F113 inocula therefore had a large effect in short term experiments and residual perturbation in long term field trials could not be detected. A good target in the effective use of a rhizosphere based biocontrol agent is during seed germination and early seedling growth especially with diseasesli ke Pythium. This is an important attribute in terms of risk assessmenat s residual effects are not desired especially in terms of the biosafety or containment of such an inoculum. An experiment was conducted to determine whether the impact of a Pseudomonas fluorescens SBW25 strain, genetically modified for kanamycin resistance and lactose utilisation, could be enhanced by soil amendment with lactose and kanamycin. Lactose addition decreased the shoot root ratio of pea and the inoculated GMM, both soil amendments increased the populations of total culturable bacteria. Lactose amendment increasedt he acid & alkaline phosphataset,h e phosphodiesterasaec tivity and the carbon cycle enzyme activities, whereas the kanamycin addition only affected the alkaline phosphatasea nd phosphodiesterasea ctivities. None of the soil enzyme activities were affected by the GMM under any of the soil amendments. The community structure with the GMM inocula in thepresence of kanamycin showed the only impact of the GMM compared to the wild type inocula. The shift towards K strategy (slower growing organisms), found in the other kanamycin amended treatments, was reduced. The functional consequenceso f the perturbation to the bacterial community profile caused by the GMM under kanamycin amended conditions is unclear, and requires further investigation, especially into the strain's efficacy as a biocontrol agent. The methodology allowed a suite of enzyme activities to be measured, enabling simultaneous evaluation of enzymes from different nutrient cycles. The measurement of a range of enzymes from various cycles allows comparative assessment of the nutritional status of the ecosystem The experimental approach incorporated varying levels of ecosystem complexity in terms of environmental control. The simplest system was the homogenised soil semi-enclosed microcosms of chapter 3 and 5 utilising environmental chambers. A higher level of microcosm complexity was used for the experiment of chapter 2 involving large intact soil cores in a glasshouse environment. The most complex system was the field trial of chapter 4, where little control over environmental conditions could be exerted. Effects of GMM inoculation were found in the least complex system, where homogenised soil of the semi-enclosed system and strictenvironmental control were applied. The more complex large intact core microcosms also proved to be a useful tool in environmental impact assessment, revealing a number of significant effects. This microcosm is therefore an effective intermediary step between the simple microcosms described previously or plant pot experiments and full scale field trials Transfer of the techniques and methodology to full scale field trials highlighted the presented problems. The inherent field variability that is a feature of most complex experiments and is beyond human control proved to be an important factor influencing the soil enzyme activities. The experiments described indicated that microbial inoculation can cause significant perturbations that can be detected by the soil enzyme methods described
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Gentically Modified Micro-organisms Molecular biology Cytology Genetics Soil science