Phylogenetic analysis of microbial diversity in the rhizoplane of oilseed rape (Brassica napus cv. Westar) employing cultivation-dependent and cultivation-independent approaches

The structure of the microbial rhizoplane community of the important crop p lant oilseed rape was studied by using a culture-dependent as well as a cul ture-independent approach based on 16S rDNA amplification. After isolation of the microbial community from the rhizoplane of oilseed rape (Brassica na pus cv. Westar), the collected suspension was divided into two parts. One p art was used for cultivation of bacteria onto three different growth media to establish a culture collection. From the other part of the rhizoplane su spension, genomic DNA was isolated and purified. Thereafter, 16S rDNA was a mplified by PCR and cloned to obtain a library of 16S rDNA genes representa tive for the bacterial communities of this habitat. Phylogenetic 16S rDNA s equence analysis of 103 clones of this library revealed considerable differ ences from the corresponding nucleotide sequences of 111 cultured bacteria. Whereas the 16S rDNA clone library was dominated by alpha -Proteobacteria and bacteria of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum (51% and 30%, respectively), less than 17% of the cultured bacteria belonged to these two groups. More than 64% of the cultivated isolates were allocated t o the beta- and gamma -subclasses of the Proteobacteria, which were present in the clone library at about 14%. Most of the clones of the alpha -Proteo bacteria of the library showed highest similarity to Bradyrhizobium sp. No such bacteria were found in the culture collection. Similarly, the second d ominant group of the clone library comprising members of the CFB phylum was represented in the culture collection by a single isolate. The phylogeneti c analysis of isolates of the culture collection clearly emphasized the nee d to use different growth media for recovery of rhizoplane bacteria. Wherea s most of the alpha -Proteobacteria were recovered on complex medium, most of the beta -Proteobacteria were isolated onto minimal media. Our results d emonstrate that the combined approach pursued in this paper is necessary to explore the biodiversity of bacterial rhizoplane communities.