Unraveling the Rhizosphere Using thecpn60 Genomic Marker and Pyrosequencing

The microbial communities of two distinct soils and of the rhizosphere, tuber-associated soil, and washed roots of potato plants grown in each soil were profiled by cpn60 gene-targeted metagenomics. DNA samples extracted from these sources were used as templates for PCR amplification of the cpn60 universal target regions present in each metagenomic sample. The cpn60 amplicons were analyzed by pyrosequencing. The 914,932 sequence reads obtained were aligned and assembled into unique cpn60 nucleotide sequences in an autonomous process that did not refer to a database of known cpn60 sequences. This process identified 27,222 unique nucleotide sequences, corresponding to 21,396 unique peptide sequences. The closest matches for each of these sequences in a database of cpn60 sequences, cpnDB, were determined. Bulk soil microbial richness [i.e., total number of unique operational taxonomic units (OTU)] was much greater than that of the plant-associated samples, as expected. The richness of the microbial communities associated with the plant samples ranged from 13% to 44% of that of the bulk soil in which it was grown. When only distinct peptide sequences derived from the nucleotide sequence OTU) were included, the apparent richness was reduced for all samples. If only OTU with higher relative abundances in the plant-associated sample than those in the bulk soil were considered, the apparent richness of the plant-associated microbial communities was significantly reduced. Clustering analysis identified OTU with distributions among the samples that strongly suggested a functional relationship with the plant. Classification of the reads observed in each sample the taxonomic level of bacterial Order revealed major differences between bulk soil and plant-associated communities. The distributions of a small number of OTU between samples suggest that these organisms had privileged relationships with the plant.