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.