S U M M A R YThe arbuscular mycorrhizal (AM) association between fungi in the order Glomales and the roots of a very wide range of vascular plants is of global ecological significance hut has proved particularly intractable to study in the field. We have developed a reliable technique to identify the fungal symbionts in roots taken directly from natural communities. Selective Enrichment of Amplified DNA combines the use of recently-developed specific DNA primers with a novel method based on the principle of subtractive hybridization to remove interfering plantderived DNA after amplification with the polymerase chain reaction. Using this technique we have shown that cndomycorrhizas of bluebells {Hyaciiithoides non-scripta) sampled directly from a woodland habitat are multispecies communities of varying composition which contain at least three genera of mycorrhizal fungi. The technique works well on a range of plant species and should have wide application to the identification of other symbionts, including pathogens. A spore survey has indicated that two particular AM types are associated with bluebells and this observation corroborates the molecular data. The presence of a Glomus species in bluebell roots was not expected from the spore data.

This study compared different methods of direct DNA extraction and purification from a silt loam soil and investigated the relationship between DNA quantity and sequence diversity. Five extraction methods and four purification techniques were investigated. Quantities of DNA extracted were between 3.4+/-0.55 and 54.3+/-8.18 &mgr;g g(-1) (dry wt) of soil with OD(260)/OD(230) purity ratios between 0.80 and 1.15. Analysis of sequence diversity in all extracts was conducted using PCR-single strand conformation polymorphism (SSCP). Profiles generated using universal 16S rDNA primers (Com1/Com2) were found to be identical when used to amplify 16S rDNA extracted directly from soil. The genus Pseudomonas was targeted in order to reduce profile complexity, which was apparent when using universal 16S rDNA primers, and which hindered direct comparison of sequence diversity. A Pseudomonas culture library and non-cultured Pseudomonas 16S rDNA genes were used to provide a background count of Pseudomonas operational taxonomic units present in the soil. Cloning and sequencing of amplicons generated using a Pseudomonas-specific (Ps-for) and a universal 16S rDNA (Com2) primer, coupled with nested amplification (Com1/Com2 amplification from Ps-for/Ps-rev amplicons), used in conjunction with SSCP, revealed that environmental contaminants co-extracted with DNA, such as humic acid, significantly reduced primer specificity. SSCP was sensitive enough to reveal template bias in different primer sets. PCR-restriction fragment length-SSCP of Pseudomonas 16S rDNA amplified from soil-extracted DNA revealed distinct differences in sequence representation between extraction methods and showed that greater DNA yield is not synonymous with higher sequence diversity. We, therefore, suggest that DNA extractions from soil should be evaluated not only in terms of quantity and purity, but also in terms of the sequence diversity present. SSCP proved to be a valuable tool for the assessment of the methodologies commonly used in PCR-mediated microbial ecology studies.

Article:Clapp, J.P., Young, J.P.W. orcid.org/0000-0001- 5259-4830 and Fitter, A.H. (1999) Ribosomal small subunit sequence diversity of Scutellospora within single spores and roots of bluebell from a woodland community. AbstractRoots of bluebell (Hyacinthoides nonscripta) were sampled from a woodland in Yorkshire, UK and spores of an arbuscular mycorrhizal fungus Scutellospora sp., were obtained from the surrounding soil. Partial small subunit (SSU) ribosomal RNA sequences were amplified from both roots and spores using either the universal forward primer SS38 or the Glomales-specific primer VANS1, with the reverse Gigasporaceaespecific primer VAGIGA. Amplified products were cloned and sequenced. Both spores and roots yielded sequences related to those known from fungi within the Glomales, with up to four distinct SSU sequences obtained from individual spores. The VANS1 primer-binding site varied considerably in sequence and only a subset of Scutellospora sequences were amplified when the VANS1 primer was used. In addition to glomalean sequences, a number of different sequences, apparently from ascomycetes, were obtained from both root and spore samples.

Summary• High levels of variation are reported in the large subunit (LSU) rRNA gene, D2 region of Glomus coronatum , a well characterized species of arbuscular mycorrhizal fungus (AMF).• Clones (435) containing the D2 regions from 7 isolates of G. coronatum were investigated for intra-and inter-isolate sequence variation using PCR-single-strand conformational polymorphism (PCR-SSCP) as a prescreen before sequencing. Isolates of G. mosseae , G. constrictum and G. geosporum , three species of AMF with similar spore ontogeny and morphology, were also analysed.• Analysis of 138 representative sequences indicated that most were unique; this variation could not be attributed to DNA polymerase or cloning artefacts. Only 13 sequences were found in more than one isolate. Neighbour-joining analysis showed that most sequences from G. coronatum formed a main group although several sequences from G. mosseae and G. constrictum clustered with G. coronatum .• There was greater than expected variation in the LSU D2 region sequences from G. coronatum . The four Glomus species, closely related by spore morphology, might represent part of a genetic continuum. Implications for the concept of species in AMF, the use of rRNA sequences to estimate biodiversity and in situ detection in field ecology are discussed.

SUMMARYThe mi'corrhizal symbiosis formed between plant roots and the arbuscular mycorrhizal (AM) fungi or Glomales is of great interest to ecologists because of its potential influence on ecosystem processes, its role in determining plant diversity m natural communities and the ability of the fungi to induce a wide variety of growth responses in coexisting plant species. Little attention, however, has been paid to the ecological role of diversity of AM fungi. Difficulties in identification, the inability to grow the fungi in pure culture, problems of taxonomic classification, and a lack of basic information on the life histories of AM fungi hinder studies of the ecological significance of diversity of .AM fungi. Nucleic acid based techniques have the potential to fill this gap in our knowledge by offering better means of identification and the opportunity to study links between the genetic diversity of AM fungi and functional and morphological diversity. The application of genus-specific molecular markers has shown that different genera of AM fungi coexist in plant roots and that this is a common occurrence. Molecular techniques have alsO' shown that natural AM fungai populations exhibit unexpectedly high genetic diversity, despite the assumption that diversity in these seemingly asexual fungi should be low. The high diversity occurs in multicopy tibosomal genes and their internal transcribed spacers, which are normally well conserved and homogeneous within an individual organLsm. The results show that sequence heterogeneity of the ribosomal genes can occur even in single spores of AM fungi, and we discuss how genetic diversity m.ay be promoted and maintained. Contrasting results, indicating that genetic diversity among replicate spores from pot-cultured material is low (even though they contain within spore sequence heterogeneity), suggest that there are mechanisms which promote high genetic diversity of AM fungi in natural ecosystems.We propose that AM fungi could be heterokaryotic as a result of the exchange of nuclei following hyphal fusion with other individuals but that other mechanisms, such as gene turnover and molecular drive, might also explain the generation of high genetic diversity without any exchange of genetic material among individuals. The high diversity in ribosomal gene sequences in AM fungi might cause problems in their use as molecular markers in field studies. \ better understanding of the levels of genetic diversity of ribosomal genes within spores, among spores of the same morphology, and among spores of differing morphology is essential to the development of sound molecular markers for field studies and to the development of a phylogenetic classification.We concltide that an understanding of tbe mechanisms which promote and maintain genetic diversity in the AM iungi is crucial, not only to further advances in ecological and evolutionary studies but also to studies of the Tiolecular basis of the regulation of the symbiosis. Moreover, we predict that while observational investigations m ....