Accurate quantification and detection of Septoria glycines in soybean using quantitative PCR

Septoria brown spot, caused by Septoria glycines, is a highly prevalent foliar disease of soybean in the United States. Accurately identifying and quantifying the pathogen in soybean can provide valuable information for disease management. In this study, we describe the development and validation of a quantitative PCR (qPCR) method to detect and quantify Septoria glycines accurately. Three sets of primers and assays were designed based on the polymorphic regions on the β-tubulin, the calmodulin, and the actin genes of S. glycines. Assays designed with the actin gene (Ac) were specific to S. glycines for both conventional PCR and qPCR. The assay designed with the β-tubulin (Bt) gene was specific to S. glycines only on the qPCR. The qPCR reaction efficiency of the Ac and Bt assays was 95 % and 98 %, respectively. The sensitivity of both Ac and Bt assays was 10 pg of S. glycines gDNA. The Bt assay was validated with field samples that had different necrotic areas. Symptoms of necrosis ranging from 0 to 30 % were significant and positively correlated (r = 0.87) to the S. glycines gDNA. The S. glycines gDNA was detected as early as 1-day post-inoculation in detached leaf assays. We expect that the assays reported here could be used for disease diagnosis and to better characterize the infection process of S. glycines.