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A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach for the detection and characterization of arbuscular mycorrhizal fungi (AMF) 18S ribosomal DNA (rDNA) was developed and applied to the study of AMF communities associated with the main sand-stabilizing plant species of the Dutch sand dunes, marram grass (Ammophila arenaria, L.). DNA was extracted directly from plant roots, soil or isolated AMF spores, and prominent bands resulting from AMF-specific DGGE profiles were excised for sequence analysis. This strategy provided a robust means of detecting and identifying AMF-like species without the use of trap plant cultivation methods. A number of Glomus-like and Scutellospora-like sequences was detected, including a putatively novel Glomus species, and differences were observed in the dominant AMF-like populations detected in healthy vs. degenerating stands of A. arenaria and in bulk sand dune soil. It has previously been suggested that plant pathogens, such as fungi and nematodes, may contribute to the decline of A. arenaria. Although no causal relationship can be drawn between the observed differences in the dominantly detected AMF-like populations and the vitality of plant growth, these results indicate that mutualistic interactions between this plant and AMF should not be overlooked when examining the role of soil-borne microorganisms in vegetation dynamics. In addition, there were discrepancies observed between the AMF-like groups detected in spore populations vs. direct 18S rDNA analysis of root material, corroborating previous suggestions that spore inspection alone may poorly represent actual AMF population structure.

In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.