Specific primers for the rapid detection and quantitation of Rhizobium elite strains of common beans in the plant and environment

Abstract

Tools for adequately detecting and monitoring elite strains used for common bean inoculation are lacking. These tools help evaluate strain competitiveness and nodule occupancy rates and study their ecology in the environment. For this reason, we designed and tested specific primers for three Rhizobium elite strains widely used as common bean inoculants: Rhizobium tropici CIAT 899, R. tropici CPAC H12, and R. freirei PRF 81. Primer specificity was confirmed in PCR assays performed with the target strains and 45 strains of the Rhizobiaceae family. CIAT 899 and CPAC H12 showed an average nucleotide identity of 99.99 %; thus, all further evaluations were performed with CIAT 899 and PRF 81. Primer amplification efficiency, sensitivity, determination coefficients, and dissociation curves were evaluated. In addition, three pairs of primers for each target strain were evaluated for their capacities to quantify their targets in the nodules, rhizosphere, and roots. Eleven and seven strain-specific primer pairs were selected for CIAT 899 and PRF 81, respectively. They amplified DNA isolated from bacterial suspensions, nodules, and nodule extracts used directly in PCR with average amplification efficiencies between 91 % and 105 % and 96 % and 107 % for CIAT 899 and PRF 81 primers and detection limits of 32 and 34 copies of target DNA, respectively. Their use showed that populations increased in the roots and decreased in the rhizospheric soil over 15 days. Nodular occupancy rates were above 90 % when the strains were inoculated individually, and a high co-infection rate was observed upon co-inoculation, with CIAT 899 and PRF 81 detected in 100 % and 73 % of the nodules, respectively. We conclude that the primers and methodologies shown here are suitable for detecting the target strains with high specificity and sensitivity. Our approach also demonstrated that R. tropici CIAT 899 and R. freirei PRF 81 were competitive and effective in infecting common beans under the tested conditions.