Sandra Caul

A Comparison of Soil Microbial Community Structure, Protozoa and Nematodes in Field Plots of Conventional and Genetically Modified Maize Expressing the Bacillus thuringiens is CryIAb Toxin

Field trials were established at three European sites (Denmark, Eastern France, South-West France) of genetically modified maize (Zea mays L.) expressing the CryIAb Bacillus thuringiensis toxin (Bt), the near-isogenic non-Bt cultivar, another conventional maize cultivar and grass. Soil from Denmark was sampled at sowing (May) and harvest (October) over two years (2002, 2003); from E France at harvest 2002, sowing and harvest 2003; and from SW France at sowing and harvest 2003. Samples were analysed for microbial community structure (2003 samples only) by community-level physiological-profiling (CLPP) and phospholipid fatty acid analysis (PLFA), and protozoa and nematodes in all samples. Individual differences within a site resulted from: greater nematode numbers under grass than maize on three occasions; different nematode populations under the conventional maize cultivars once; and two occasions when there was a reduced protozoan population under Bt maize compared to non-Bt maize. Microbial community structure within the sites only varied with grass compared to maize, with one occurrence of CLPP varying between maize cultivars (Bt versus a conventional cultivar). An overall comparison of Bt versus non-Bt maize across all three sites only revealed differences for nematodes, with a smaller population under the Bt maize. Nematode community structure was different at each site and the Bt effect was not confined to specific nematode taxa. The effect of the Bt maize was small and within the normal variation expected in these agricultural systems.

Migration of bacterial-feeding nematodes, but not protozoa, to decomposing grass residues

SummaryPopulations of bacterial-feeding nematodes and protozoa developing in soil amended with dried grass powder or a nutrient solution were monitored in experimental systems designed to prevent migration from surrounding unamended soil. The addition of nutrient solution stimulated both microbial activity, as determined by dehydrogenase activity, and protozoa, but brought about no increase in nematode numbers. Amendment of soil with grass, however, caused an increase in both types of grazer, with the maximum biomass of protozoa (180 μg g-1) exceeding that of bacterial-feeding nematodes (42 μ g-1). The decomposing grass was rapidly colonised by rhaditid nematodes, mainly Caenorhabditis sp. Incubating grass-amended soil in the absence of any surrounding soil, to prevent migration, changed the microflora from predominantly bacterial to predominantly fungal, and so could not be used to compare treatments with and without migration. Surrounding the amended soil with sterilised soil prevented migration and caused no detectable change in the microflora. This treatment demonstrated that migration plays an important part in the colonisation of decomposing substrates by nematodes, but that protozoa do not migrate in soil. The nematodes migrated from a volume of unamended soil that was equivalent to eight times the volume of amended soil. The potential effects of the large grazing pressure on the subsequent decomposition of the grass residue are discussed.

Soil microbial and faunal responses to herbicide tolerant maize and herbicide in two soils

A glasshouse experiment was set up to compare processes and organisms in two soils planted with genetically modified (GM) herbicide tolerant (HT) maize treated with appropriate herbicides. This was part of a wider project (ECOGEN) looking at the consequences of GM cropping systems on soil biology using a tiered approach at laboratory, glasshouse and field scales. Soil for the experiment was taken from field sites where the same maize cultivars were grown to allow comparison between results under glasshouse and field conditions. The maize cultivars T25 (GM HT glufosinate-ammonium tolerant), Orient (non HT near isogenic control for T25) and Monumental (a conventional, non HT variety) were grown in contrasting sandy loam and clay loam soils, half were sprayed with the appropriate herbicide as used in the field and soil samples were taken at the five-leaf and flowering plant growth stage. The main effects on all measured parameters were those of soil type and plant growth stage, with four categories of subsequent interaction: (1) there were no effects of herbicide on plant growth or soil microarthropods: (2) the maize cultivar (but not the GM HT trait) had effects on the decomposition of cotton strips and the nematode community; (3) herbicide application in general altered the community level physiological profile of the microbial community and reduced both soil basal respiration and the abundance of protozoa; and (4) the specific application of glufosinate-ammonium to T25 maize altered soil microbial community structure measured by ester linked fatty acids. The results from this glasshouse experiment support the findings from the field that there are effects of herbicide application on the soil microbial and meso-faunal community but that, compared to other standard agricultural practices, the differences are relatively small.

Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference.

Application of polymerase chain reaction (PCR) techniques has developed significantly from a qualitative technology to include powerful quantitative technologies, including real-time PCR, which are regularly used for detection and quantification of nucleic acids in many settings, including community analysis where culture-based techniques are not suitable. Many applications of real-time PCR involve absolute quantification which is susceptible to inaccuracies caused by losses during DNA extraction or inhibition caused by co-extracted compounds. We present here an improvement to this approach involving the addition of an artificial internal standard, prior to nucleic acid extraction. The standard was generated by in-situ mutagenesis from an E. coli template to ensure it both did not amplify with bacterial primers used for quantification and was short enough to minimise possible interference with other analyses. By estimating gene target copies by relative abundance, this approach accounts for both loss during extraction and inhibition effects. We present a novel application of relative real time PCR, using the internal standard as a reference, allowing accurate estimation of total bacterial populations both within and across a wide range of soils and demonstrate its improvement over absolute quantification by comparison of both approaches to ester linked fatty acid analysis of the same soils.

Growth of a ciliate protozoan in model ballotini systems of different particle sizes

The effect of structure (i.e. particle size) on protozoan population development was studied using liquid culture, containing known amounts of the bacterium Erwinia cartovora subsp. carotovora or nutrient growth medium PPY, and the ciliate protozoan Tetrahymena pyriformis. Structure was introduced into each system in the form of different size ranges of ballotini (glass beads), or sand. Even with the smallest particle sizes used, all pore pathways were accessible to protozoa. Incorporation of structure into nutrient solution acted to lower significantly (P < 0.05) protozoan activity in the structured pore network, as compared to the nutrient solution without structure. There were no significant differences (at the 5% level) in the final protozoa population in either substrate system. As particle size decreased, the protozoan population also decreased. Structure was shown to introduce distances between protozoa and bacterial cells thus, in comparison with treatments without structure, protozoan populations were significantly reduced. Further, reducing particle size would increase the time taken to explore the available pore volume, and reduce the amount of food available in each pore. The outcome was that decreasing the particle size reduced the feeding rate and so reduced the rate of population increase. Other possible physical mechanisms which may limit protozoan movement, such as surface area, are examined.

Early root herbivory impairs arbuscular mycorrhizal fungal colonization and shifts defence allocation in establishing Plantago lanceolata

Research into plant-mediated indirect interactions between arbuscular mycorrhizal (AM) fungi and insect herbivores has focussed on those between plant shoots and above-ground herbivores, despite the fact that only below-ground herbivores share the same part of the host plant as AM fungi. Using Plantago lanceolata L., we aimed to characterise how early root herbivory by the vine weevil (Otiorhynchus sulcatus F.) affected subsequent colonization by AM fungi (Glomus spp.) and determine how the two affected plant growth and defensive chemistry. We exposed four week old P. lanceolata to root herbivory and AM fungi using a 2×2 factorial design (and quantified subsequent effects on plant biomass and iridoid glycosides (IGs) concentrations. Otiorhynchus sulcatus reduced root growth by c. 64%, whereas plant growth was unaffected by AM fungi. Root herbivory reduced extent of AM fungal colonization (by c. 61%). O. sulcatus did not influence overall IG concentrations, but caused qualitative shifts in root and shoot IGs, specifically increasing the proportion of the more toxic catalpol. These changes may reflect defensive allocation in the plant against further attack. This study demonstrates that very early root herbivory during plant development can shape future patterns of AM fungal colonization and influence defensive allocation in the plant.