Molecular approaches for taxa discovery in plant-associated soil microbial communities

N. ROSENZWEIG, J. Kang, L. L. Kinkel, J. M. Bradeen

Department of Plant Pathology, University of Minnesota, St. Paul, MN, USA

Phytopathology 98:S136

Plants influence rhizosphere microbial community composition, but there is little systematic information on the variation in microbial communities associated with different plant hosts. This work explores the challenges and limitations of current molecular techniques in resolving microbial community structure. Soil cores (n = 10 for each plant species) were collected from the rhizosphere of vigorous and well-established Andropogon gerardii (Ag) and Lespedeza capitata (Lc) plants at the Cedar Creek Ecosystem Science Reserve in east-central Minnesota. Ribosomal 16S V3 fragments from rhizosphere bacterial communities were studied using denaturing gradient gel electrophoresis (DGGE) and targeted sequencing. DGGE patterns were unable to distinguish microbial communities associated with environmental samples from each plant species. To evaluate the sensitivity of DGGE, ‘communities’ of varying complexity were simulated by mixing different bacterial clones. Only slight DGGE banding differences were observed between phylogenetically distinct taxa. Targeted sequencing of the 16S V3 region revealed differences in microbial communities associated with Ag and Lc, though the data are limited by the small sample sizes possible with traditional cloning approaches. These results indicate significant limitations in the use of DGGE for resolution of complex microbial communities. As an alternative, we are developing a novel resource of diversity-enriched clone libraries and pursuing large-scale sequencing of 16S V3 region fragments. Our combined approaches will yield a resource-efficient means of characterizing complex environmental samples and discovering rare and under-represented taxa.