Notas Científicas Amplification of 16 S rRNA gene sequences to differentiate two highly related bradyrhizobia species

A 16S rRNA gene PCR-based assay was developed aiming at a fast molecular diagnostic method to differentiate the two phylogenetically closely related species Bradyrhizobium japonicum and B. elkanii, isolated from soybean nodules, in order to identify those more competitive and comprising greater nitrogen fixation ability for use in the formulation of commercial inoculants. The assay used was able to discriminate ten reference strains belonging to these two Bradyrhizobium species, as well as to efficiently identify 37 strains isolated from fields cultivated with soybean.

The massive inoculation of Brazilian soils comprising few bradyrhizobia strains belonging to B. japonicum and B. elkanii species has resulted in an established population in most soils cropped with this leguminous (Ferreira & Hungria, 2002).However, these populations may show morphological, biochemical, physiological, genetic and symbiotic variability related to adaptation processes and genetic transfer (Hungria & Vargas, 2000;Ferreira & Hungria, 2002).Literature data have shown that these new bacterial populations differed significantly from the original introduced strains (Ferreira & Hungria, 2002), including a high percentage of nodules occupied by strains with unknown serological reaction after some years of soybean cropping (Freire et al., 1983).
The fact that B. japonicum and B. elkanii species are largely used together in Brazilian soybean fields makes sometimes practically impossible to differentiate strains isolated from soybean nodules.This problem is particularly important when the objective of the screening is to identify strains more competitive and comprising greater nitrogen fixation ability to be used in the formulation of the commercial inoculants.
In this work, the development of a PCR-based assay targeting the 16S rRNA gene, which was able to discriminate the two related B. japonicum and B. elkanii species among 10 reference strains and to identify 37 strains isolated from fields cultivated with soybean is reported.Genomic DNA from these bacteria was also used in RFLP analysis of 16S-23S rDNA ITS and nifH gene regions to confirm their taxonomic classification.
The  (Somasegaran & Hoben, 1994) for seven days at 28 o C. Agrobacterium tumefaciens AGL1, Escherichia coli XL1-Blue, Azospirillum brasilense Sp7 ATCC 2914 and Paenibacillus macerans ATCC8244 were cultivated in LB medium (Sambrook & Russel, 2001) for 24 hours at 37ºC.Fifty microliters of the liquid cultures were heated in a boiling water bath.The supernatants obtained after a brief spin were used as a DNA template.
To determinate the most suitable annealing temperature, PCR reactions were performed using a temperature gradient varying from 45 to 64ºC, in which the temperature of 55ºC presented better amplification results.All final PCR amplifications were performed in a Thermo Hybaid thermal cycler using an initial denaturation step at 94ºC for 1 min, 30 cycles of 1 min of denaturation at 94ºC, 30 s of annealing at 55ºC, 1 min of extension at 72ºC, followed by a final extension for 5 min at 72ºC.The amplification products were electrophoresed in 1% agarose gel containing ethidium bromide in 1 X Tris-EDTA buffer (TBE) and the gels were photographed under UV light.
For the 16S-23S rDNA ITS spacer RFLP fingerprinting primers pHr (Massol-Deya et al., 1995) and p23S uni322anti (Honeycutt et al., 1995) were used.Fragments of nifH gene were amplified using the primers PolF and PolR defined by Poly et al. (2001) to amplify a 360 base pair nifH fragment from DNA isolated from a wide range of diazotrophic microorganisms.PCR were conducted as described above.Ten microliters of the purified amplification products were overnight digested using MboI and HaeIII endonucleases (Promega Life Sciences) for ITS region and HaeIII and SalI endonucleases (Promega Life Sciences) for nifH gene fragment.The restriction fragments were separated on 8% acrylamide-bisacrylamide gel stained with silver nitrate (Sambrook & Russel, 2001).
The specificity of the PCR system developed in this work was confirmed by the application of the primer pairs (Brady + Belk and Brady + Bjap) to amplify DNA obtained from cultures of well-characterized Bradyrhizobium species, which are traditionally recommended as commercial inoculants in Brazilian fields (Figure 1 A).The reference strains were also subjected to the RFLP analysis using the molecular markers 16S-23S rDNA ITS spacer, as suggested by Willems et al. (2003), and the nifH gene, since its phylogeny has been reported to be largely consistent with the 16S rDNA tree (Zehr et al., 2003).The RLFP analysis of the nifH gene was able to discriminate strains belonging to B. japonicum or B. elkanii species, corroborating the results obtained with the Bradyrhizobium-specific PCR approach.However, the RFLP analysis of the 16S-23S rDNA ITS spacer presented variable patterns within the same specie, and could not discriminate these two bradyrhizobia species.
Group-specific primers developed in this work were also used with DNA obtained from other bacterial strains available in the laboratory: Rhizobium leguminosarum bv.trifolii, Agrobacterium tumefaciens AGL1, Escherichia coli XL1-Blue, Azospirillum brasilense Sp7 ATCC 2914 and Paenibacillus macerans ATCC8244, as well as with DNA obtained from two other Bradyrhizobium species, B. betae PL7HG1 and B. canariense BTA-1.These bacteria were used as negative controls to check the specificity of the primer pairs developed.Either no PCR products were obtained or DNA fragments bigger than 400 bp were yielded (Figure 1 B and C).
After the confirmation of the PCR specificity with the reference strains, the method was used to identify isolates obtained from fields cultivated with soybean.From a total of 37 isolates, 24 were shown as belonging to the B. japonicum and 13 as belonging to the B. elkanii species (Figure 1 B and C).These 37 isolates were also subjected to the RFLP analysis using the molecular markers 16S-23S rDNA ITS spacer and the nifH gene.Exactly the same results to those obtained with the reference strains were achieved.
The assay was efficient in discriminating Bradyrhizobium field isolates, and proved to be of fast and relatively low cost.These features can be useful when the objective is to characterize genotipically bradyrhizobia species for agricultural practices.