Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA.

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

Spatial structure in soil chemical and microbiological properties in an upland grassland.

We characterised the spatial structure of soil microbial communities in an unimproved grazed upland grassland in the Scottish Borders. A range of soil chemical parameters, cultivable microbes, protozoa, nematodes, phospholipid fatty acid (PLFA) profiles, community-level physiological profiles (CLPP), intra-radical arbuscular mycorrhizal community structure, and eubacterial, actinomycete, pseudomonad and ammonia-oxidiser 16S rRNA gene profiles, assessed by denaturing gradient gel electrophoresis (DGGE) were quantified. The botanical composition of the vegetation associated with each soil sample was also determined. Geostatistical analysis of the data revealed a gamut of spatial dependency with diverse semivariograms being apparent, ranging from pure nugget, linear and non-linear forms. Spatial autocorrelation generally accounted for 40-60% of the total variance of those properties where such autocorrelation was apparent, but accounted for 97% in the case of nitrate-N. Geostatistical ranges extending from approximately 0.6-6 m were detected, dispersed throughout both chemical and biological properties. CLPP data tended to be associated with ranges greater than 4.5 m. There was no relationship between physical distance in the field and genetic similarity based on DGGE profiles. However, analysis of samples taken as close as 1 cm apart within a subset of cores suggested some spatial dependency in community DNA-DGGE parameters below an 8 cm scale. Spatial correlation between the properties was generally weak, with some exceptions such as between microbial biomass C and total N and C. There was evidence for scale-dependence in the relationships between properties. PLFA and CLPP profiling showed some association with vegetation composition, but DGGE profiling did not. There was considerably stronger association between notional sheep urine patches, denoted by soil nutrient status, and many of the properties. These data demonstrate extreme spatial variation in community-level microbiological properties in upland grasslands, and that despite considerable numeric ranges in the majority of properties, overarching controlling factors were not apparent.

Broad-scale analysis of soil microbial community DNA from Upland grasslands.

We have applied a broad-scale approach to the analysis of DNA extracted from soils which support characteristic grasslands at an upland site in the UK. To test for the degree of coherence between microbial and vascular communities, grasslands were characterised as 'improved', 'semi-improved', or 'unimproved', according to the degree of management they had received and consequent botanical composition. Microbial DNA was extracted directly from the grassland soils and analysed by three techniques: (i) thermal denaturation, which profiles the guanine and cytosine (G + C) base distribution within the community; (ii) cross hybridisation of the DNA which measures the degree of similarity between the samples; (iii) measurement of reassociation kinetics of denatured DNA, which provides a measure of the complexity of the DNA. Thermal denaturation revealed significant differences in the %G + C composition of the communities. DNA from the improved soil had the highest median %G + C value, whilst that from the unimproved soil had the lowest. The relative distribution of G + C bases also differed significantly between the samples from the three grasslands. Cross hybridisation of DNA from the different soils also indicated significant differences in the degree of similarity between the DNA from the grasslands, with unimproved showing 59% similarity to improved. Indices from the cross hybridisation assay suggested that, in terms of complexity, the samples ranked unimproved > semi-improved > improved. Reassociation kinetics supported this conclusion, but the rates of reassociation were such that less than 40% reassociation occurred over a 31-day period, thus preventing calculation of C(o)t1/2.

Direct extraction of microbial community DNA from humified upland soils.

This paper describes a protocol effective at extracting high yields of high-purity microbial community DNA from humified soils. DNA was extracted from soil by lysozyme, SDS and freeze-thaw lysis, precipitated and then subjected to a double caesium chloride density gradient centrifugation stage before concentrating and washing. Evaluation using three soils yielded up to 30 micrograms DNA g-1 dry soil, with absorbance ratios at 260:230 nm and 260:280 nm of 1.6-2.0. The DNA extracted from the three soils was digested by four restriction enzymes and a 16S rDNA eubacterial product was amplified by PCR. These tests indicated that the DNA obtained by the protocol was sufficiently pure for molecular biological analysis.

Scanning electron microscopy of the gut microflora of two earthworms: Lumbricus terrestris and Octolasion cyaneum.

Scanning electron microscopy was used to investigate the presence of microorganisms, probably bacteria, on the gut surface of earthworms. The washed surfaces of the intestines of two earthworms, Lumbricus terrestris and Octolasion cyaneum, were examined. Numerous organisms resembling bacteria were observed throughout the gut, some in situations suggesting attachment. Compared with similar investigations in other invertebrates, there were fewer bacteria, showing less morphological diversity, on the earthworm gut surface. The majority of organisms viewed were coccoid, some were filamentous, and a few rod-shaped cells were observed. Cocci, often in chains, were seen in the foregut of both species. Although cocci were also numerous in the midgut region, particularly in the typhlosole, in O. cyaneum tufts of segmented, filamentous organisms were also seen with some segments resembling spores. Fewer organisms were found in the hindgut, but in L. terrestris there were segmented, filamentous organisms, attached to the epithelium by way of a "socket-like" structure, similar to that by which segmented, filamentous bacteria (SFBs) are attached to the ileum of rats and mice. Transmission electron microscopy of the hindgut of L. terrestris was undertaken to explore the structure and attachment of SFBs to the gut epithelium. However, although a few rod-shaped bacteria were observed, no SFBs were located. The observations reported here provide evidence that earthworms have an attached gut microflora of filamentous microorganisms which are probably indigenous, and as far as we are aware this is the first published report of such findings in these invertebrates.