Desenvolvimento e validação de método para o monitoramento de rizóbios elite inoculados no feijoeiro utilizando PCR e qPCR

Abstract

The common bean performs symbiosis with rhizobia, so Biological Nitrogen Fixation is a sustainable alternative to nitrogen fertilizers. However, the success of the symbiosis depends on several biotic and abiotic factors, among them the competitiveness of selected strains against the natural microbiota in the soil. One way to assess this competitiveness is by evaluating the nodular occupation and persistence of the bacteria in the soil. Unfortunately, the currently existing methods are laborious and/or do not have sufficient specificity to differentiate the inoculated strains of soil native rhizobia. Therefore, the Polymerase Chain Reaction (PCR) and its variants are presented as a convenient and highly specific alternative for the monitoring and quantifying of microorganisms associated with plants and/or in the soil. In this context, the objective of the present work was to design and validate strain-specific primers to detect and quantify the bacteria Rhizobium tropici strain CIAT 899 and CPAC H12 and R. freirei strain PRF 81, using PCR and Real-Time PCR (qPCR). To this end, the primers were designed from coding regions of the genome and selected in silico to contain DNA sequences unique to the genome of each strain. The validation process of these primers involved exclusive specificity testing for the target strains in relation to others genetically close and distant to them, PCR assays involving several DNA extracts, and evaluation of nodular occupancy in greenhouse experiments. First, each primer pair was evaluated for its amplification efficiency and dissociation curve. Then, the best pairs were used for quantifying target strains from DNA samples isolated from roots and rhizospheric soil. Finally, the primers were tested for their applicability to evaluate the nodular occupation rate and quantify the elite rhizobia population in rhizospheric soil and common bean roots under field conditions. This information allowed us to discuss the nodulation and the performance of the rhizobial-plant interaction, as well as the fitness of strains CIAT 899, CPAC H12, and PRF 81 to nodulate the common bean.