Dazzo, F.B., M.J. Klug, E. Peters, M. Cole, J.M. Tiedje, S. Marsh, and G.P. Robertson
Presented at the All Scientist Meeting (1999-07-20 to 1999-07-21 )
In general, nutrient enrichments from seedling exudation select for zymogenous microorganisms that colonize the rhizoplane of plant roots grown in soil. However, most microbial ecology studies of the rhizoplane allow living roots to develop in soil for at least 4-5 days before sampling since it is generally though that very young roots are not sufficiently developed to select a microbial community. Indeed, little is known of the very first community of microorganisms that colonize roots grown in soil. The goal of this project is therefore to define the community ecology of the pioneer rhizobacteria that colonize roots of white clover (a nodulated, endomycorrhizal legume) and spring canola (a non-nodulated, non-mycorrhizal brassica) during the first 2 days of germination in a sandy loam soil from an LTER site at KBS. These rhizoplane communities are compared to the pioneer community that colonizes toothpicks (a non-viable woody substratum control) buried in the same soil.Plating experiments on 10% standard plate count/cyclohexamide agar and soil extract/glucose/cyclohexamide agar indicate that the culturable pioneer communities (CFU/mg root dry wt) colonize each of the 3 substrata with similar, initial doubling rates (~ 3 hrs doubling time). However, the duration of exponential growth was 3-5 times longer on the true rhizoplanes, resulting in 103-104-fold higher community densities at 2 days, consistent with their greater resource allocation.Cluster analyses of quantitative cellular FAME data from 626 numerically dominant culturable isolates indicated 600 distinct FAME profiles, only 4% of which clustered. Thus, the pioneer rhizobacterial communities that developed on white clover, canola, and toothpicks were very diverse, contrary to the ecological characteristic that pioneer communities generally have low diversity dominated by just a few species. Further cluster analyses indicated that the structures of each of these pioneer culturable communities were distinct and changed over the 2 day period. A subset of 106 isolates is being analyzed by ARDRA, BIOLOG, and FISH (using group-specific 16SrRNA fluorescent probes) to further assess the diversity, nutritional versatility and phylogeny of these culturable pioneer communities. In addition, T-RFLP analyses are being done on whole rhizoplane communities of clover, canola, and toothpicks sampled at 1 & 2 days. These ongoing tests should further indicate the dynamics and diversity of microbial communities during pioneer colonization of roots, their extent of habitat-selectivity, evidence of ecological succession during pioneer colonization of the rhizoplane, and which bacteria first colonize roots in soil.We also plan to perform computer-assisted microscopy to learn more about the in situ spatial relationships of pioneer rhizobacterial colonization of roots growing in soil. Two major innovative developments have been made to facilitate this work. First, we have developed improved protocols to dislodge soil from roots so that >80% of the rhizoplane can be visualized by scanning electron microscopy and laser scanning confocal microscopy. Second, we have developed new computer-assisted microscopy software, called CMEIAS© (for Center for Microbial Ecology Image Analysis System), tailored to studies of in situ community microbial ecology. These will be used to quantitate the morphological diversity, abundance (numbers, biovolume, biomass C, biosurface area), and spatial relationships (spatial density, % cover, nearest neighbor distribution, and centroid x,y coordinates to compute semivariogram autocorrelation and krieging density maps via GS+ Geostatistics) of the pioneer rhizobacterial communities developing on roots grown in soil. This information will significantly enhance our understanding of the diversity, dynamics and ecological succession of pioneer rhizobacterial communities on a spatial scale relevant to the microorganisms themselves.
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