Multiplex and quantitative PCR targeting SCAR markers for strain-level detection and quantification of biofertilizers

Biofertilizers are the eco-friendly bio-input being used to sustain the agriculture by reducing the chemical inputs and improving the soil health. Quality is the major concern of biofertilizer technology which often leads to poor performance in the field and thereby loses the farmers' faith. To authenticate the strain as well as its presumed cell load of a commercial product, sequence characterized amplified region (SCAR) markers were developed for three biofertilizer strains viz., Azospirillum brasilense (Sp7), Bacillus megaterium (Pb1) and Azotobacter chroococcum (Ac1). We evaluated the feasibility of multiplex-PCR and quantitative real-time PCR for SCAR marker-based quality assessment of the product as well as the persistence of the strains during crop growth. We showed that multiplex PCR can concurrently discriminate the strains based on the amplicons' size and detects up to 10⁴ cells per g or per ml of carrier-based or liquid formulation of biofertilizer, respectively. The detection limit of quantitative PCR targeting SCAR markers is 10³  cells per g or ml of biofertilizer. Both the PCR methods detected and quantified them in the maize rhizosphere. Hence SCAR marker-based quality assessment would be a sensitive tool to monitor the biofertilizer production as well as its persistence in the inoculated crop rhizosphere.