Quantitative estimation of flagellate community structure and diversity in soil samples.

Heterotrophic flagellates occur in nearly all soils and, in most cases, many different species are present. Nevertheless, quantitative data on their community structure and diversity are sparse, possibly due to a lack of suitable techniques. Previous studies have tended to focus on either total flagellate numbers and biomass, or the identification and description of flagellate species present. With the increased awareness of the role of biodiversity and of food web interactions, the quantification of species within the community and their response to environmental change is likely to become more important. The present paper describes a modification of the most probable number method that allows such a quantification of individual flagellate morphotypes in soil samples. Observations were also made on the biomass of flagellate morphotypes in soil. 20 to 25 morphotypes of heterotrophic flagellates were detectable per gram of two different arable soils, which were treated experimentally to test the technique. One of the soils was fumigated with chloroform vapour for different lengths of time (0, 0.5, 2 or 24 hours); this led to a reduction in the number of morphotypes, in the Shannon diversity index and in the evenness. The other soil was planted with wheat, and while rhizosphere soils contained the same morphotypes as bulk soil, the abundance of individual morphotypes was significantly different and the Shannon diversity index in rhizosphere soils was significantly higher. Soil influenced by an elevated CO2 level likewise differed significantly in morphotype abundance when compared to soil exposed to ambient levels of CO2. The technique recovered more than 80% of the discernible morphotypes and could also be used to quantify amoebal and ciliate communities in a similar way.

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.

The effect of nitrate-nitrogen supply on bacteria and bacterial-feeding fauna in the rhizosphere of different grass species.

Microbial growth in the rhizosphere is affected by the release of organic material from roots, so differences in carbon budgets between plants may affect their rhizosphere biology. This was tested by sampling populations of bacteria and bacteriophagous fauna from the rhizosphere of Lolium perenne, Festuca arundinacea, Poa annua, and Poa pratensis, under conditions of high and low nitrate availability. Concentrations of soluble phenolics and lignin varied considerably between the species but were not related to differences in rhizosphere biology. L. perenne and F. arundinacea supported fewer bacteria than the Poa species. There was no significant rhizosphere effect on the groups of protozoa. The major indicators of rhizosphere productivity were the bacterial-feeding nematodes (mainly Acrobeloides spp.), and there was a large positive effect of added nitrate. Nematode biomass was significantly lower in the rhizosphere of the slow-growing P. pratensis compared with the fast-growing P. annua, indicating that the differential allocation of carbon has affects on rhizosphere biology. A large rhizosphere effect on enchytraeid worms was also observed, and their potential importance in the rhizosphere is discussed.

Does the Presence of Detached Root Border Cells of Zea mays Alter the Activity of the Pathogenic Nematode Meloidogyne incognita?

ABSTRACT The root-knot nematode Meloidogyne incognita is a major pathogen of a range of important crops. Currently, control is typically achieved by the use of nematicides. However, recent work suggests that manipulating the ability of roots to slough off border cells, which then act as a decoy to the nematode, can significantly decrease damage to the roots. We investigated the attractiveness of border cells to M. incognita and the response of the nematode to border cells in close proximity. We found very limited attraction, in that nematodes did not preferentially alter direction to move toward the border cells, but a large and significant increase in nematode speed was observed once they were in the immediate vicinity of border cells. We discuss the results in the context of physical and biological mechanisms in relation to the control of pathogenic nematodes.

Morphological and Histochemical Changes Occurring during the Life-span of Root-tip Galls on Lolium perenne Induced by Longidorus elongatus.

The RNA and protein content of perennial ryegrass root-tip galls induced by Longidorus elongatus were measured from transverse sections and the morphology described. Galls progressed through five distinct stages and were viable for only 10-12 days at 18 C, after which they collapsed and became necrotic. In the initial stage hypertrophy occurred and cells contained enlarged nuclei and nucleoli, a greater proportion of cytoplasm, and increased concentrations of protein. This was followed by hyperplasia; cells divided to give two or four daughter cells, accompanied by a proportionate reduction in volumes of cytoplasm, nuclei, and nucleoli and reduced concentrations of RNA and protein. The third stage was secondary hypertrophy with enlarged, amoeboid nuclei and nucleoli and a significant increase in concentration of RNA and protein. In the final two stages, as feeding by L. elongatus progressively removed cell contents, most cells were devoid of inclusions and galls collapsed and were invaded by soil bacteria. This ordered development and exploitation of galls suggests that L. elongatus may have two phases in its feeding.

Nuclear changes induced by the nematode Xiphinema diversicaudatum in root-tips of strawberry.

Feeding by the nematode X. diversicaudatum caused a progressive increase in the DNA content and size of strawberry nuclei. After four days feeding, nuclei had DNA values intermediate between 8C and 16C and had increased in size from a mean of 17 micron2 for control root tips to 49 micron2. Multinucleate cells were present after two and four days feeding. There were no ultrastructural differences in the composition of nuclei from control and parasitized root tips, but strawberry nuclei consisted mainly of dispersed chromatin whereas ryegrass nuclei contained a large proportion of condensed chromatin.

Nuclear changes induced by the nematode Longidorus elongatus in root-tips of ryegrass, Lolium perenne.

The plant ectoparasitic nematode Longidorus elongatus induces distinct changes in the root-tip nuclei of perennial ryegrass (Lolium perenne L.). There was an initial shift in DNA content into the 4C category, followed by further increases with values intermediate between 8C and 16C present after eight days. Nuclear size was reduced after six days due to increased nuclear division during hyperplasia. Nuclear DNA content and size decreased in galls older than eight days as a result of nuclear disintegration induced by L. elongatus. In cells near to the feeding site, nuclei became split into several, small globules. In more distant nuclei DNA-containing material became dispersed throughout the cytoplasm. In the oldest galls, cells were largely devoid of nuclei.

Nuclear changes induced by the nematodes Xiphinema diversicaudatum and Longidorus elongatus in root-tips of perennial ryegrass, Lolium perenne.

The DNA content and size of individual nuclei from galls of perennial ryegrass root-tips induced by X. diversicaudatum and L. elongatus were measured. Feeding by X. diversicaudatum increased the DNA content of the nuclei by varying amounts. No regular doubling pattern of the DNA content was discernible. The DNA values varied up to between 32-64C. Generally the size of the nuclei was not increased, although some were larger than control nuclei. The modified nuclei probably have an altered metabolic function, which increases the food value of the gall to the nematode. Some bi-nucleate cells were also observed, which probably result from mitosis without cytokinesis. A preliminary examination of nuclei from galls induced by L. elongatus revealed similar nuclear changes, but no bi-nucleate cells were found.

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.

The impact of bacterial diet on the migration and navigation of Caenorhabditis elegans.

Can diet have a significant impact on the ability of organisms to sense and locate food? Focusing on the bacterial feeder Caenorhabditis elegans, we investigated what effect preconditioning on a range of bacterial substrates had on the subsequent chemotaxis process involved in the nematode locating other bacterial populations. Remarkably, we found that C. elegans, initially fed on a diet of Escherichia coli OP50, was significantly impaired in finding E. coli OP50 populations, compared to other available bacterial populations (P <0.001). We found similar results for another bacterial feeding nematode species, suggesting that a general "substrate legacy" may operate across a wide range of organisms. We discuss this important finding with respect to the variation in response exhibited within a given nematode population, and the impact nematode migration has on bacterial dispersal in the environment.

The relationship between microbial community structure and functional stability, tested experimentally in an upland pasture soil.

Soil collected from an upland pasture was manipulated experimentally in ways shown previously to alter microbial community structure. One set of soil was subjected to chloroform fumigation for 0, 0.5, 2, or 24 h and the other was sterilised by gamma-irradiation and inoculated with a 10(-2), 10(-4), 10(-6), or 10(-8) dilution of a soil suspension prepared from unsterilized soil. Following incubation for 8 months, to allow for the stabilization of microbial biomass and activity, the resulting microbial community structure (determined by PCR-DGGE of bacterial specific amplification products of total soil DNA) was assessed. In addition, the functional stability (defined here as the resistance and resilience of short-term decomposition of plant residues to a transient heat or a persistent copper perturbation) was determined. Changes in the active bacterial population following perturbation (determined by RT-PCR-DGGE of total soil RNA) were also monitored. The manipulations resulted in distinct shifts in microbial community structure as shown by PCR-DGGE profiles, but no significant decreases in the number of bands. These shifts in microbial community structure were associated with a reduction in functional stability. The clear correlation between altered microbial community structure and functional stability observed in this upland pasture soil was not evident when the same protocols were applied to soils in other studies. RT-PCR-DGGE profiles only detected a shift in the active bacterial population following heat, but not copper, perturbation. We conclude that the functional stability of decomposition is related to specific components of the microbial community.