Structural and functional characterization of bacterial diversity in the rhizospheres of three grain legumes

The aim of this study was to increase the understanding of diversity and activity of dominant bacterial populations in the rhizospheres of three economically important grain legumes (Vicia faba, Lupinus albus and Pisum sativum). A cultivation-independent approach was employed to achieve this aim bearing in mind the limitation of cultivation-dependent technique that only 10% of bacteria present in rhizosphere can be cultured. PCR amplification of 16S rDNA and subsequent separation of the amplicons by DGGE was used in an initial screening of replicates for experimental variation and for the first characterization of bacterial community composition of the three rhizospheres under study. Specific profiles generated by the three legumes, derived by both 16S rDNA and rRNA, emphasized the need to perform detailed analysis of the communities present in these rhizospheres. Clone libraries for PCR and RT-PCR products were generated for representative samples of all the three legumes. Firmicutes were found to be the most dominant in all the legumes, both in DNA- and RNA-derived libraries, indicating them to be the most active group as well. A plant-dependent rhizosphere effect was reflected by the absence of ? -subdivision members in Pisum and ? -subdivision members of proteobacteria in Vicia rhizosphere. High numbers of as yet unclassified bacteria were also obtained. With this experimental set-up, using the same soil material but three different legumes and a uniform inoculation with Rhizobium sp. , it became evident that plant roots influence the development of bacterial communities in the rhizosphere in a plant-specific manner. The extent of the rhizosphere effect could vary in natural field conditions as the present study was performed under controlled conditions in green house using soil from agricultural site. Extraction and analysis of rRNA has enabled identification of active taxa in the present study. Fingerprints were obtained for total RNA using two different primers. The profiles generated revealed marked differences between the three rhizospheres of the three legumes under study, indicating differences between the metabolic status of the bacterial communities present in the rhizospheres of these three legumes. To address the question of functional diversity, mRNA extraction and subsequent RT-PCR were performed for various genes important in nutrient cycling. The presence of chitinase genes could be established by specific PCR amplification using DNA extracted from the three rhizospheres. However, no expression of the gene could be detected by RT-PCR. Enzyme assays confirmed no or very low levels of the chitinase protein in the rhizospheres. Analysis of proteolytic enzymes (serine and neutral metallopeptidases) showed the presence and activity of serine peptidase in the three rhizospheres. Neutral metallopeptidase gene was also present in the three rhizospheres but no expression could be detected in the Lupinus rhizosphere. This was a confirmation of plant-dependent effect at the level of functioning of the bacterial communities. Genes for nitrite reductase (nirK and nirS), which may lead to removal of nitrogen from the system by denitrification, were targeted to gain an understanding of the importance of this enzyme in a nitrogen-enriching environment. The presence of nirS was not detected in any of the legume rhizospheres, but both the presence and activity of nirK was established for the three rhizospheres. The diversity of this gene was investigated by generating clone libraries with the RT-PCR products from the three plant rhizospheres. The observation of distinct differences in the distribution of phylotypes of expressed nirK gene in the three legume rhizospheres confirmed a plant specific effect on the functions of the rhizosphere bacterial communities. The present study revealed a hitherto unknown diversity of rhizospheric bacteria associated with grain legumes. Entirely cultivation-independent approaches to characterize the structure and function of the bacterial community of the rhizosphere of the three grain legumes clearly revealed plant-dependent rhizosphere effect on bacterial community structure and function.