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

Abstract

-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.Index terms: PCR, rhizobial diversity, commercial inoculants. Amplificação de seqüências do gene RNAr 16S para diferenciar duas espécies de bradirrizóbios altamente relacionadasResumo -Foi avaliado um método baseado em PCR do gene do RNAr 16S, desenvolvido com a finalidade de um diagnóstico molecular rápido na identificação das espécies filogeneticamente relacionadas Bradyrhizobium japonicum e B. elkanii, isoladas de nódulos de soja, visando ao seu emprego na identificação de estirpes mais competitivas e com maior capacidade de fixação de nitrogênio para uso na formulação de inoculantes comerciais. O método usado foi capaz de discriminar dez estirpes-padrão pertencentes a essas duas espécies de Bradyrhizobium, assim como identificar eficientemente 37 estirpes isoladas de lavouras cultivadas com soja.Termos para indexação: PCR, diversidade de rizóbios, inoculantes comerciais.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 Pesq. agropec. bras., Brasília, v.42, n.9, p.1361-1364, set. 2007 A. Giongo et al. 1362 was also use...

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“…Total DNA was extracted from pure cultures as described by Giongo et al (2007). Selected primers PolF and PolR (Poly et al 2001) were used to amplify a 360-bp region of nifH as described previously ).…”