Isolation and 16S rRNA sequence analysis of the beneficial bacteria from the rhizosphere of rice.

The present study deals with the isolation of plant growth promoting rhizobacteria (PGPR) from rice (variety NIAB IRRI-9) and the beneficial effects of these inoculants on two Basmati rice varieties. Nitrogen-fixing activity (acetylene-reduction activity) was detected in the roots and submerged shoots of field-grown rice variety NIAB IRRI-9. Estimation of the population size of diazotrophic bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) in roots and shoots indicated about 10(5)-10(6) counts/g dry weight at panicle initiation and grain filling stages. Four bacterial isolates from rice roots and shoots were obtained in pure culture which produced phytohormone indoleacetic acid (IAA) in the growth medium. Among these, three isolates S1, S4, and R3 reduced acetylene to ethylene in nitrogen-free semi-solid medium. Morphological and physiological characteristics of the isolates indicated that three nitrogen-fixing isolates S1, S4, and R3 belonged to the genus Enterobacter, while the non-fixing isolate R8 belonged to the genus Aeromonas. 16S rRNA sequence of one isolate from root (R8) and one isolate from shoot (S1) was obtained which confirmed identification of the isolates as Aeromonas veronii and Enterobacter cloacae, respectively. The 1517-nucleotide-long sequence of the isolate R8 showed 99% similarity with Aeromonas veronii (accession No. AF099023) while partial 16S rRNA sequence (two stretches of total 1271 nucleotide length) of S1 showed 97% similarity with the sequence of Enterobacter cloacae (accession No. AJ251469). The seedlings of two rice varieties Basmati 385 and Super Basmati were inoculated with the four bacterial isolates from rice and one Azospirillum brasilense strain Wb3, which was isolated from wheat. In the rice variety Basmati 385, maximum increase in root area and plant biomass was obtained in plants inoculated with Enterobacter S1 and Azospirillum Wb3, whereas in the rice variety Super Basmati, inoculation with Enterobacter R3 resulted in maximum increase of root area and plant biomass. Nitrogen fixation was quantified by using 15N isotopic dilution method. Maximum fixation was observed in Basmati 385 with the inoculants Azospirillum Wb3 and Enterobacter S1 where nearly 46% and 41% of the nitrogen was derived from atmosphere (%Ndfa), respectively. In general, higher nitrogen fixation was observed in variety Basmati 385 than in Super Basmati, and different bacterial strains were found more effective as inoculants for the rice varieties Basmati 385 and Super Basmati.

Burkholderia graminis sp. nov., a rhizospheric Burkholderia species, and reassessment of [Pseudomonas] phenazinium, [Pseudomonas] pyrrocinia and [Pseudomonas] glathei as Burkholderia.

In a survey of soil and wheat or maize rhizoplane bacteria isolated using a medium containing azelaic acid and tryptamine as sole carbon and nitrogen sources, respectively, a large proportion of Burkholderia-like bacteria were found. Among them, a homogeneous group of strains was identifiable based on phenotypic properties, fatty acid composition, DNA-DNA hybridizations and 16S rDNA sequences. According to molecular data, this group belongs to the genus Burkholderia but its weak similarity to previously described species suggests that it belongs to a novel species. Closest 16S rDNA phylogenetic neighbours of this species are Burkholderia caryophylli and two previously named Pseudomonas species which clearly appear to be part of the Burkholderia genus and were thus named Burkholderia glathei comb. nov. and Burkholderia phenazinium comb. nov. Strains of the new species are oxidase- and catalase-positive, produce indole and gelatinase, and use L-xylose, lactose, rhamnose, trehalose, D-lyxose, L-arabitol, xylitol and D-raffinose as sole carbon source. This novel taxon is named Burkholderia graminis. In the course of this study, [Pseudomonas] pyrrocinia also proved to be a member of the Burkholderia genus.

Oligonucleotide probes based on 16S rRNA sequences for the identification of four Azospirillum species.

Partial sequences of the 16S rRNA molecules of nine strains belonging to four Azospirillum species were used to design species-specific oligonucleotide probes. Azospirillum strains sequences were analyzed and three homologous fragments containing 16 nucleotides were determined. These three probes were found to be characteristic of A. lipoferum (Al), A. irakense (Ai), and A. brasilense/amazonense species (Aba) and of few nontarget organisms. The specificity of these three probes was tested both against sequences in the GenBank data base and in numerous colony hybridization experiments. As a few non-target organisms hyridized with the different Azospirillum probes, the use of these probes in bulk soil hybridization is not permitted. However, their use together with specific isolation techniques is validated.

Molecular relationship of an atypical Azospirillum strain 4T to other Azospirillum species.

DNA/DNA hybridization, plasmid content and partial 16S rDNA sequence were determined to confirm the relationship between two Azospirillum strains, 4B and 4T, isolated from the same rice rhizosphere. The partial 16S rDNA sequence was determined for 9 strains belonging to 5 Azospirillum species which included Azospirillum lipoferum strains 4B and 4T, and was compared to a set of ribosomal sequences from other bacteria of the alpha subdivision of the Proteobacteria. Four Azospirillum species sequences were found to form a coherent group, whereas A. halopraeferens was found to be more divergent. The five Azospirillum species were closely related to Magnetospirillum and to a lesser extent to Rhodospirillum, with which they share some morphological features. The partial 16S rDNA sequences of the two strains 4B and 4T were identical and confirmed the closeness of these two strains. Plasmid content and DNA/DNA hybridization data (S1 nuclease and delta Tm) grouped Azospirillum strains 4B and 4T with the other strains of species Azospirillum lipoferum (USA5a, 59b, BR17). Other biochemical and genetic characters confirmed the grouping of Azospirillum strains 4B and 4T together with A. lipoferum.