Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes

Abstract

Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa. The classification of the root-and stem-nodulating bacteria of legumes formerly based mainly on symbiotic characteristics is in constant evolution. Since 1988 two new genera, Sinorhizobium and Azorhizobium, have been proposed (3, 7). The number of species within the genus Rhizobium increased from four to eight with the addition of Rhizobium tropici (29), R. galegae (27), R. huakuii (4), and R. etli (37) to the previously recognized species, R. meliloti, R. leguminosarum, R. loti (20), and R. fredii (35). Within the genus Bradyrhizobium, a new species of slow-growing soybean microsymbionts, Bradyrhizobium elkanii, has been proposed in addition to B. japonicum (22). However, other groups of J?Jizobium strains which do not cluster with the existing species have been identified. This is the case for tree rhizobia (15, 45), group B of R. meliloti (9), and two Rhizobium genomic species (11, 24) and some other genotypes nodulating Phaseolus vulgaris (8, 10, 29, 32). As more knowledge is acquired and new isolates are studied, new species are discovered and the former species are split. Consequently, more and more leguminous plants (Glycine, Lupinus, and Lotus spp. [20]) appear to be nodulated by rhizobia belonging to different genera or to different Rhizobium and Bradyrhizobium species. For instance, three species, R. leguminosarum bv. phaseoli, R. etli bv. phaseoli, and R. tropici, two new Rhizobium genomic species (24), and other unclassified genotypes (29, 32) have been isolated from nodules of P. vulgans. The ability of legumes to be nodulated by several Rhizobium species might well appear to be the rule rather than the exception. Symbiotic performance, which has been the most practical trait, although not absolute, for differentiation between species is thus less and less usable. There is therefore a more and more pressing need for an easy and rapid method which would enable rhizobiologists, and especially ecologists, to identify the rhizobia they are working with.

Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco

“…Gel electrophoresis of undigested PCR products revealed that all tested strains produced a single band of about 1500 bp. This size corresponded well to the expected size of the 16S rDNA genes of most members of the Rhizobiaceae (Laguerre et al 1994).…”

Biodiversity of Mimosa pudica rhizobial symbionts (Cupriavidus taiwanensis, Rhizobium mesoamericanum) in New Caledonia and their adaptation to heavy metal-rich soils

“…PCR-restriction fragment length polymorphism (RFLP) analysis was performed using 10 lL of 16S rRNA gene PCR amplification products obtained with CfoI, HinfI and MspI restriction enzymes. Analysis of digestion products by agarose gel electrophoresis was performed as previously described (Laguerre et al, 1994). PCR amplification of recA fragments of 800 bp were obtained for beta-rhizobia strains using primers recABurk1F and recA-Burk1R, and fragments of 600 bp were obtained for alpha-rhizobia using primers recA-6-F and TS2recAR (Table 2).…”

Plant growth-promoting Burkholderia species isolated from annual ryegrass in Portuguese soils

“…Amplification and sequence analysis of the 16S rRNA, recA and nifH genes Near full-length 16S rRNA gene (ca. 1Á5 kb) was amplified with the universal primers fD1 and rD1 (Weisburg et al 1991) from extracted genomic DNA using the PCR procedures described by Laguerre et al (1994). A fragment of the recA gene (869 bp) was amplified using the Burkholderia-specific primers BUR1 and BUR2 described by Payne et al (2005).…”