RESUMO
In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5' domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the beta subclass of the class Proteobacteria, the Cytophaga-Flexibacter-Bacteriodes group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition.
Assuntos
Bactérias/classificação , Ecossistema , Microbiologia do Solo , Bactérias/química , Bactérias/genética , Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana , DNA Bacteriano/análise , DNA Bacteriano/genética , Ácidos Graxos/análise , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Little is known about Ceanothus-infective Frankia strains because no Frankia strains that can reinfect the host plants have been isolated from Ceonothus spp. Therefore, we studied the diversity of the Ceonothus-infective Frankia strains by using molecular techniques. Frankia strains inhabiting root nodules of nine Ceanothus species were characterized. The Ceanothus species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of Frankia strains that infect Ceanothus spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3' end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among the Ceanothus-infective Frankia strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the Ceanothus host species. Instead, the Frankia strains present were related to the sample collection locales.
Assuntos
Actinomycetales/classificação , Actinomycetales/genética , Variação Genética , Plantas/microbiologia , DNA Bacteriano/genética , Genes de RNAr , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Estados UnidosRESUMO
Molecular phylogenetic trees were reconstructed from nucleotide sequences of nifH and 16S rDNA for Frankia and of rbcL for actinorhizal plants. Comparison of Frankia phylogenetic trees reconstructed using nifH and 16S rDNA sequences indicated that subgroupings of both trees correspond with each other in terms of plant origins of Frankia strains. The results suggested that 16S rDNAs can be utilized for coevolution analysis of actinorhizal symbioses. Frankia and plant phylogenetic trees reconstructed using 16S rDNA and rbcL sequences were compared. The comparison by tree matching and likelihood ratio tests indicated that although branching orders of both trees do not strictly correspond with each other, subgroupings of Frankia and their host plants correspond with each other in terms of symbiotic partnership. Estimated divergence times among Frankia and plant clades indicated that Frankia clades diverged more recently than plant clades. Taken together, actinorhizal symbioses originated more than three times after the four plant clades diverged.