A ribosomal RNA gene intergenic spacer based PCR and DGGE fingerprinting method for the analysis of specific rhizobial communities in soil.
A direct molecular method for assessing the diversity of specific populations of rhizobia in soil, based on nested PCR amplification of 16S-23S ribosomal RNA gene (rDNA) intergenic spacer (IGS) sequences, was developed. Initial generic amplification of bacterial rDNA IGS sequences from soil DNA was followed by specific amplification of (1) sequences affiliated with Rhizobium leguminosarum "sensu lato" and (2) R. tropici. Using analysis of the amplified sequences in clone libraries obtained on the basis of soil DNA, this two-sided method was shown to be very specific for rhizobial subpopulations in soil. It was then further validated as a direct fingerprinting tool of the target rhizobia based on denaturing gradient gel electrophoresis (DGGE). The PCR-DGGE approach was applied to soils from fields in Brazil cultivated with common bean (Phaseolus vulgaris) under conventional or no-tillage practices. The community fingerprints obtained allowed the direct analysis of the respective rhizobial community structures in soil samples from the two contrasting agricultural practices. Data obtained with both primer sets revealed clustering of the community structures of the target rhizobial types along treatment. Moreover, the DGGE profiles obtained with the R. tropici primer set indicated that the abundance and diversity of these organisms were favoured under NT practices. These results suggest that the R. leguminosarum-as well as R. tropici-targeted IGS-based nested PCR and DGGE are useful tools for monitoring the effect of agricultural practices on these and related rhizobial subpopulations in soils.
de Oliveira
Valéria Maia
VM
Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas- CPQBA/UNICAMP, CP 6171, CEP 13081-970, Campinas, SP, Brazil.
Manfio
Gilson Paulo
GP
da Costa Coutinho
Heitor Luiz
HL
Keijzer-Wolters
Anneke Christina
AC
van Elsas
Jan Dirk
JD
D000383:Agriculture / Q000379:methods; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D016961:Rhizobium leguminosarum / Q000235:genetics / Q000254:growth & development*; D042162:Rhizobium tropici / Q000235:genetics / Q000254:growth & development*; D016415:Sequence Alignment; D012988:Soil Microbiology
D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer