Genotypic Characterization of Bradyrhizobium Strains Nodulating Small Senegalese Legumes by 16S-23S rRNA Intergenic Gene Spacers and Amplified Fragment Length Polymorphism Fingerprint Analyses

Abstract

We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.Due to their nitrogen-fixing symbiosis with soil bacteria collectively named rhizobia, legumes play an important role in the nitrogen cycle, especially in the tropics. They are used to restore or increase soil fertility of degraded soils in intercropping systems, as green manure, and to produce medicinal and commercial by-products. In Senegal (West Africa), many native legumes are important plant resources for many purposes, and they are the only alternatives to the costly and pollutive mineral nitrogen fertilizers used in agriculture. Many of these legumes are well adapted to the local arid climatic conditions. As drought and soil erosion progress, they are still present as colonizers. The family Leguminosae is considered to be of tropical or subtropical origin, and many of the recently proposed new rhizobial species originate from leguminous plants from these zones that had not been previously investigated.After several prospections around the country, we focused on 27 native nodulated leguminous plants species belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, Zornia, which play an important ecological role and have agronomic potential in arid regions. As very little or no information concerning their associated rhizobia were available so far, we obtained 71 nodule isolates from these legumes in different regions in Senegal and first characterized them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell protein profiles and by 16S amplified ribosomal DNA restriction analysis (ARDRA) fing...

RFLP analysis of the rRNA operon of a Brazilian collection of bradyrhizobial strains from 33 legume species

“…S2). As a comparison, when bradyrhizobia nodulating Senegalese legumes were analysed by PCR-RFLP of the IGS with eight enzymes, the strains were clustered at a final level of similarity of 36 % (Doignon-Bourcier et al, 2000).…”

“…The fragments produced with the primers were larger than those obtained by Willems et al (2001) and Doignon-Bourcier et al (2000), but we have used different primers. Cluster analysis of the RFLP products of the IGS resulted in two main great groups and isolated strains that were joined at a final level of similarity of 26?7 % (Table 1; Supplementary Fig.…”

“…S2). As a comparison, when bradyrhizobia nodulating Senegalese legumes were analysed by PCR-RFLP of the IGS with eight enzymes, the strains were clustered at a final level of similarity of 36 % (Doignon-Bourcier et al, 2000).When variability in the 16S rRNA gene sequence is low, it has also been suggested that analysis of the 23S rRNA region can be useful, as the longer fragment contains more information with a faster rate of sequence change (Olsen & Woese, 1993;Ludwig & Schleifer, 1994;Wang & Martínez-Romero, 2000). Indeed, in our study, analysis of this region resulted in the largest number of differences and the strains were grouped at a very low level of similarity (16 %).…”

Molecular diversity, effectiveness and competitiveness of indigenous rhizobial population infecting mungbean Vigna radiata (L. Wilczek) under semi-arid conditions

“…It was observed that individual groups of rhizobia were present at one site and rhizobia belonging to cluster 2 or 4 were present at all the sites. In case of Bradyrhizobium infecting tree legume, bradyrhizobial strains were clustered into 16 groups with six predominant groups [34]. Further symbiotic characterization of these rhizobia showed that mungbean rhizobia differed in number of nodules formed, nodule fresh weight, root dry weight, shoot dry weight and total N uptake by plant.…”

Genomic diversity of Astragalus cicer microsymbionts revealed by AFLP fingerprinting

“…It plays also a big role in a taxonomy of microorganisms including classification and typing of nodule bacteria (Doignon-Bourcier et al, 2000;Duim et al, 2004;Gibson et al, 1998;Gürtler and Mayall, 2001;Savelkoul et al, 1999;Terefework et al, 2001).…”