Development of real-time PCR assay for detection and quantification ofSinorhizobium melilotiin soil and plant tissue

Abstract

Sinorhizobium meliloti is a nitrogen-fixing alpha-proteobacterium present in soil and symbiotically associated with root nodules of leguminous plants. To date, estimation of bacterial titres in soil is achieved by most-probable-number assays based on the number of nodules on the roots of test plants. Here, we report the development of two real-time PCR (qPCR) assays to detect the presence of S. meliloti in soil and plant tissues by targeting, in a species-specific fashion, the chromosomal gene rpoE1 and the pSymA gene nodC. rpoE1 and nodC primer pairs were tested on DNA extracted from soil samples unspiked and spiked with known titres of S. meliloti and from plant root samples nodulated with S. meliloti. Results obtained were well in agreement with viable titres of S. meliloti cells estimated in the same samples. The developed qPCR assays appear to be enough sensitive, precise and species-specific to be used as a complementary tool for S. meliloti titre estimation. These two novel markers offer the possibility of quick and reliable estimation of S. meliloti titres in soil and plant roots contributing new tools to explore S. meliloti biology and ecology including viable but nonculturable fraction.