Field response of rice paddy crop to Azospirillum inoculation: physiology of rhizosphere bacterial communities and the genetic diversity of endophytic bacteria in different parts of the plants

The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated. A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA. The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation. The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library. The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48%. Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring. The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated. The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes. The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community. The diversity within the archaeal domain was low. Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity). The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea. The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.