Applied Soil Ecology 14 2000 125–134
G+C profiling and cross hybridisation of microbial DNA reveals great variation in below-ground community structure in UK upland grasslands
Christopher D. Clegg
∗
, Karl Ritz, Bryan S. Griffiths
Soil Plant Dynamics Unit, Division of Plant, Soils and Environment, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Received 27 July 1999; received in revised form 21 January 2000; accepted 26 January 2000
Abstract
Total DNA was directly extracted from microbial populations in grassland soils taken from three geographically distinct upland sites at Garrigill, Aber and Sourhope, UK. Within each site, grasslands were categorised using the National Vegetation
Classification into distinct vegetation sequences, namely unimproved, semi-improved and improved. Microbial community DNA was extracted from the different soils and analysed by determining percent guanine+yctosine G+C profiles by
thermal denaturation, and by cross hybridisation to measure the degree of similarity between the DNA extracted from the different soils. The G+C profiles indicated that the microbial community structure within the different grasslands at Garrigill
was significantly different. No significant differences in G+C were detected under the different grasslands at Aber and Sourhope. However, significant differences in G+C profiles derived from spatially-distinct replicate quadrats taken within
grasslands were detected within the semi-improved grasslands at each site, and the unimproved grassland at Aber. Cross hybridisation analysis revealed significant differences between the improved, semi-improved and unimproved grasslands
within all sites, with similarity values ranging from 51 to 94. Significant differences were also detected between replicate quadrats within grassland types by this technique. These results provide evidence for great spatial variation in community
DNA i.e. genetic composition of microbial communities within these grasslands. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Community DNA; G+C profiling; Cross hybridisation; Microbial diversity; Soils; Grasslands
1. Introduction
Plant communities can often be described in terms of the characteristic species of which they are com-
posed. What is much less clear is whether this also holds with respect to the microbial communities as-
sociated with a particular vegetation type, and the degree to which microbial communities regulate plant
∗
Corresponding author. Present address: Institute of Grassland and Environmental Research, North Wyke Research Station, Oke-
hampton, Devon EX20 2SB, UK. Tel.: +44-1837-883500; fax: +
44-1837-82139. E-mail address: christopher.cleggbbsrc.ac.uk C.D. Clegg
communities and vice versa. Microorganisms exist in the soil in the form of complex communities of di-
verse composition. Factors regulating such assemblies in soils are poorly understood, although there is some
evidence that vegetation affects the density and com- position of soil communities e.g. Turkington et al.,
1988; Chanway et al., 1991; Westover et al., 1997. Mechanisms by which this occurs may be based on the
rate and quality of substrate input to soils which plays an important role in governing soil microbial com-
munity structure Griffiths et al., 1999. One of the primary sources of such substrate is material derived
from vegetation, ranging from readily-assimilable substrates exuded from the roots of actively growing
0929-139300 – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 0 0 0 0 0 4 5 - 7
126 C.D. Clegg et al. Applied Soil Ecology 14 2000 125–134
plants to more recalcitrant ligno-cellulose complexes which constitute the plants structural material. There
is variation in the composition of these components from different plant species, with bacterial selec-
tion occuring in the rhizosphere Lemanceau et al., 1995; Mahaffee and Kloepper, 1997; Siciliano et al.,
1998.
One of the difficulties in attempting to understand relationships between plant and microbial community
structure is that of description. It is relatively easy to qualify and quantify vegetation composition, whilst it
is much more difficult to analyse the composition of soil microbial communities. Traditional culture-based
measurements provide both a limited and selective representation of the total community as only 2–4 of
bacteria may be readily isolated from soil Olsen and Bakken, 1987. Phenotypic diversities of microbial
populations in soils have been profiled by measur- ing potential function using BIOLOG Garland and
Mills, 1991, phospholipid fatty acid PLFA analysis Frostegärd et al., 1996 and also fatty acid methyl
ester FAME analysis Cavigelli et al., 1995. Molec- ular techniques have also been applied to the study
of microbial populations in environmental samples. These have tended towards the application of specific
oligonucleotide probes directed at DNA Guo et al., 1997, rRNA e.g. Stahl et al., 1988; Kämpfer et al.,
1996 or to rRNA sequence analysis of microbial com- munities e.g. Giovannoni et al., 1990; Stackebrandt et
al., 1993. In a more wide ranging approach, reassoci- ation kinetics of DNA extracted from soils have sug-
gested that as many as 10,000 different bacterial types may be present in 100 g soil Torsvik et al., 1996.
Such a broad-scale approach based on the analysis of community DNA may offer a useful level of reso-
lution for plant:microbe community analyses. Com- munity DNA can be analysed by techniques which
provide different but complementary information about the overall genetic structure of the community.
These include percent guanine+cytosine G+C profiling Holben and Harris, 1995; Griffiths et al.,
1997; Clegg et al., 1998, community cross hybridis- ation Lee and Fuhrman, 1990; Griffiths et al., 1996;
Clegg et al., 1998 and reassociation kinetics Torsvik et al., 1990; Ritz et al., 1997; Clegg et al., 1998,
denaturing gradient gel electrophoresis DGGE Muyzer et al., 1993 and terminal restriction length
polymorphism Liu et al., 1997. In this paper we report on G+C profiling and
community DNA cross hybridisation to determine the genetic composition of soil microbial communities
under upland grasslands of characteristic vegetation types from three geographically distinct sites in the
UK. A central hypothesis in this study was that there is coherence between the structure of plant communi-
ties and their associated soil microbial assemblages. By comparing community structure between quadrats,
we also examined the degree of spatial variation in community structure within these grasslands.
2. Materials and Methods
