Fast and efficient symbiotic gene-based duplex PCR approach for the preliminary selection of legume root nodule bacteria

Abstract

The isolation of root nodule bacteria (RNB) usually lead to obtainment of several non-rhizobia, mainly those fast-growing bacteria. The plant-authentication experiments to select the nodulating bacteria are time consuming and can be ruined in case of nodulation of negative controls. To speed up this step in rhizobiology research a fast, simple and efficient duplex PCR assay for the amplification of nodC and nifH genes in was developed aiming to separate rhizobia and non-rhizobia in RNB culture collections. The method was optimized with 43 reference strains and was applied to a new culture collection for validation. Considering all bacteria, 106 out of 109 nodulating rhizobia were positive for both genes. Among non-nodulating, 48 out of 48 were nodC-negative. The results demonstrated the efficiency of this new method that can be completed in a day, to save time and money in RNB isolation projects, fulfilling Koch's postulate, and separating rhizobia from non-rhizobial bacteria.