C. D. Clegg

The impact of grassland management regime on the community structure of selected bacterial groups in soils

The impact of long-term grassland management regimes on microbial community structure in soils was assessed using multivariate analysis of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) banding patterns of selected bacterial groups and PLFA (phospholipid fatty acid) profiling. The management regimes assessed were inorganic nitrogen (N) fertiliser application and soil drainage. PCR-DGGE profiles of the eubacteria, actinomycetes, ammonia oxidisers and pseudomonads were assessed by principal co-ordinate analysis of similarity indices which were generated from binary data using both Dice and Jaccard coefficients. The analysis of binary DGGE data revealed significant impacts of N fertiliser on the eubacterial and actinomycete community structure using the Jaccard coefficient, whilst N fertiliser had a significant impact on the actinomycete community structure only when using similarity indices generated from the Dice coefficient. Soil drainage had a significant impact on the community structures of the actinomycetes and the pseudomonads using both Dice and Jaccard derived similarity indices. Multivariate analysis of principal components derived from PLFA profiling revealed that N fertiliser had a significant impact on the microbial community structure. Although drainage alone was not a significant factor in discriminating between PLFA community profiles of the different treatments, there was a significant interaction with N fertiliser. Analysis of principal component analysis (PCA) loadings revealed that PLFAs i15:0 and i17:0 were partly responsible for the clustering away of the undrained-N fertilised treatment. Although soil management regime influenced some background soil data, correlation analysis using PC1 from PLFA data revealed no significant relationship with soil organic matter, pH, total C and total N. These results provide evidence that grassland management practices impact on the community composition of specific microbial groups in soils.

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.

Interaction of a genetically modified Pseudomonas fluorescens with the soil-feeding earthworm Octolasion cyaneum (Lumbricidae)

Abstract The geophagous earthworm Octolasion cyaneum was maintained in microcosms for up to 14 d in the presence of a genetically-modified microorganism (GEMMO), Pseudomonas fluorescens KTG. The GEMMO contained a marker cassette, which was inserted into the chromosome, consisting of the genes coding for kanamycin and gentamycin resistance and also a cry IVB sequence. Plate counts of P. fluorescens KTG were higher in the burrow wall on day 2, and lower on days 7 and 14 than those in the unworked bulk soil. Numbers of P. fluorescens KTG were consistently significantly lower in cast material than in the unworked soil. Counts for total bacteria revealed no significant differences between bulk soil, burrow wall and casts. When earthworms were fed on soil containing P. fluorescens KTG, the population size of the GEMMO declined progressively on passage from the foregut to the hindgut, then increased slightly in the casts relative to the hindgut. However counts in fresh casts were still significantly lower than the corresponding uningested soil. Populations of P. fluorescens KTG in casts increased by up to approximately 10-fold over the first 2 d of the ageing period. Thereafter, plate counts of the GEMMO were slightly less than the corresponding soil kept under the same conditions, showing a similar rate of decline over the 50-d period. Total bacterial plate counts in the aged casts increased by approximately 25-fold during the first 2 d of incubation, subsequently declining whilst remaining significantly higher than the total bacterial plate counts in the corresponding soil which remained relatively constant throughout the experiment. Following a single exposure of the earthworms to the GEMMO, counts of the modified bacterium were detected in casts for upto 15 d. The interactions between bacteria and earthworms are discussed in relation to the potential for dispersal of GEMMOs by soil invertebrates.

Biophysical processes affecting the transit of a genetically-modified Pseudomonas fluorescens through the gut of the woodlouse Porcellio scaber

Abstract Our objective was to determine the effect of gut transit retention time of genetically-modified bacteria ingested by the woodlouse Porcellio scaber . The experimental animals were supplied ash leaf litter inoculated with the genetically-modified bacterium Pseudomonas fluorescens KTG and bacteria in food and faeces were counted using selective plating and immunofluorescent techniques. The bacteria were also detected using the polymerase chain reaction (PCR). It was found that plate counts of P. fluorescens KTG in fresh faeces were lower than those found in the litter when the GEMMO was supplied to animals at five different population densities, suggesting that a proportion of the GEMMO population was lost during gut transit. There was no significant difference in the survival of freshly cultured and starved cultures of P. fluorescens KTG on gut transit through P. scaber as determined by plate counts in fresh faeces. Retention time of P. fluorescens KTG in the woodlouse gut was found to be longer than that of the food bolus. The passage of bacteria through the gut was modelled and tracked using microbeads of a size similar to bacteria. Fluorescent microbeads added to food litter were detected within the anterior chamber, papillate region and rectum of the woodlouse for at least 17 days after ingestion. Scanning electron microscopy revealed that beads were retained within the cuticular structure of the digestive tract and also within mucopolysaccharide produced within the gut. Immunofluorescent observations of washed hindgut samples provided little evidence to suggest P. fluorescens KTG had become attached to the hindgut wall during transit. Very few colonies of the GEMMO and indigenous bacteria were detected from homogenised hepatopancreas samples. P. fluorescens KTG was however detected in the hepatopancreas of P. scaber using PCR. It is suggested that the retention of bacteria within the guts of woodlice is by physical rather than biological mechanisms such as growth or attachment.