Characterization of Rhizobium ‘hedysari’ by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting

Abstract

The taxonomic and discriminatory power of RFLP analysis of PCR amplified parts of rhizobial rrn operons was compared to those of genomic PCR fingerprinting with arbitrary and repetitive primers. For this purpose, the two methods were applied for characterization of a group of bacterial isolates referred to as Rhizobium 'hedysari'. As outgroups, representatives of the family Rhizobiaceae, belonging to the Rhizobium galegae, Rhizobium meliloti, Rhizobium leguminosarum and Agrobacterium tumefaciens species were used. By the RFLP analysis of the PCR products corresponding to the variable 5'-half of the 23S rRNA gene and of the amplified spacer region between the 16S and 23S rRNA genes all Rh. 'hedysari' strains studied were tightly clustered together while the outgroups were placed in an outer position. The PCR products of the 3' end parts of the 23S rDNA did not show significant RFL polymorphism and no species differentiation on their basis was possible. In parallel, analysis of the same strains was performed by PCR amplification of their DNA with 19, 18 and 10 bp long arbitrary primers (AP-PCR) as well as with single primers corresponding to several bacterial repetitive sequences (rep-PCR). By both AP and rep-PCR an identification of every particular strain was achieved. In general, all primers provided taxonomic results that are in agreement with the species and group assignments based on the RFLP analysis of the rrn operons. On the basis of the results presented here it can be concluded that AP and rep-PCR are more informative and discriminative than rDNA and RFLP analysis of the rhizobial strains studied.

Identification and discrimination of thiobacilli using ARDREA, RAPD and rep-APD

“…Recently, a fast and reliable procedure based on the RFLP analysis of three PCR amplified parts of the rrn operon corresponding to the intergenic spacer (IGS) between 16S and 23S rRNA-genes, and to the 3?-and 5?-ends of the 23S rDNA (ARDREA), was applied for classification and identification of natural Rhizobium isolates (Selenska-Pobell et al 1996). In the work presented here, another group of soil bacteria was analysed by ARDREA.…”

“…The PCR amplifications of the six rrn operon fragments shown in Fig. 1 were performed under the conditions described in Selenska-Pobell et al (1996). The PCR products were digested with the restriction enzymes AvaII, CfoI, DdeII, HaeIII, MspI, NdeII and RsaI (Gibco-BRL, Life Technologies, Eggenstein, Germany).…”

“…The PCR products were digested with the restriction enzymes AvaII, CfoI, DdeII, HaeIII, MspI, NdeII and RsaI (Gibco-BRL, Life Technologies, Eggenstein, Germany). The resulting patterns were analysed as described earlier (Selenska-Pobell et al 1996).…”

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

“…Moreover, PCR-restriction fragment length polymorphism (PCR-RFLP) of IGS has been reported to be a useful fingerprinting method to characterize bacterial strains, with a higher discriminating power than the 16S ARDRA method. It has been applied to known rhizobial species, such as Rhizobium leguminosarum, Rhizobium "hedysari," Sinorhizobium meliloti, and Rhizobium galegae (6,22,31,33), and also tropical rhizobia strains (18) and Bradyrhizobium strains from the Canary Islands (37).…”

Random amplified polymorphic DNA analysis of effective Rhizobium sp. associated with beans cultivated in brazilian cerrado soils

“…The RAPD technique is a polymerase chain reaction (PCR) based assay that was developed to detect polymorphisms in genomic DNA (19,20). Besides being simpler and cheaper, this method is as effective as the more labor intensive RFLP for establishing genetic relationships and identifying Rhizobium strains (3,17).…”