ABSTRACT
The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.
Subject(s)
Base Sequence , Bradyrhizobium/growth & development , Bradyrhizobium/isolation & purification , Fabaceae/growth & development , Gene Amplification , In Vitro Techniques , Polymerase Chain Reaction/methods , Rhizobium/growth & development , Rhizobium/isolation & purification , MethodsABSTRACT
The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.
ABSTRACT
AIM: To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. METHODS: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. RESULTS: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.
Subject(s)
Bacteria/classification , Plant Proteins/genetics , RNA, Ribosomal, 16S/genetics , Soil Microbiology , Bacteria/genetics , Bacteria/isolation & purification , Biomarkers , DNA, Bacterial/analysis , DNA-Directed RNA Polymerases , Ecosystem , Electrophoresis, Polyacrylamide Gel , Genetic Variation , PhylogenyABSTRACT
Pyrolysis mass spectrometry (PyMS) and DNA fingerprinting (RAPD and RSalpha hybridization) were used to characterize soybean inoculant strains and root nodule isolates of bradyrhizobia from the Brazilian Cerrado soils. Most isolates were shown to be derived from the inoculant strains on the basis of genotype comparisons by DNA fingerprinting. Phenotypic analysis (using PyMS) of the strains and separately of the polysaccharides derived from them showed that the nodule isolates differed from the parental strains, suggesting adaptation to the Cerrado soil environment. The extent of the differences between the derivatives and inoculant strains was similar for comparisons made on the basis of whole-cell preparations or from the isolated polysaccharides, indicating that the adaptation was caused by changes in the composition of the polysaccharides produced.
Subject(s)
Adaptation, Physiological , Bradyrhizobium/growth & development , Bradyrhizobium/genetics , Glycine max/microbiology , Polysaccharides, Bacterial/chemistry , Soil Microbiology , Bradyrhizobium/chemistry , Brazil , Mass Spectrometry/methods , Nucleic Acid Hybridization , Polysaccharides, Bacterial/genetics , Polysaccharides, Bacterial/metabolism , Random Amplified Polymorphic DNA TechniqueABSTRACT
A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA). A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed. Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil. The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced. The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined. Of the 124 clones sequenced, 98.4% were from the domain Bacteria. Two of the rDNA sequences were derived from eukaryotic organelles. Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence. No sequences of the domain Archaea were found. Within the domain, Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low G+C-content gram-positive group (21.8%). Some kingdoms, such as the Thermotogales, the green nonsulfur group, Fusobacteria, and the Spirochaetes, were absent. A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al. (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol., 176:1-6, 1994). From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.
Subject(s)
Soil Microbiology , Australia , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Base Sequence , Chimera , Cloning, Molecular , DNA Primers/genetics , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , DNA, Fungal/genetics , DNA, Fungal/isolation & purification , DNA, Ribosomal/genetics , DNA, Ribosomal/isolation & purification , Ecosystem , Fungi/genetics , Fungi/isolation & purification , Genetic Variation , Japan , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , WisconsinABSTRACT
The Brazilian inoculant strains 29W and 587 were found to be members of Bradyrhizobium elkanii primarily on the basis of 16S rRNA gene sequences identical to that of B. elkanii USDA76 and on the basis of reactivity with antibodies against serogroups 76 and 31, respectively. The agronomic consequences of using strains of B. elkanii as soybean inoculants are discussed.
