Development of assays to differentiate and quantify three Cercospora species causing Cercospora leaf blight on soybean

Cercospora leaf blight (CLB) is the most devastating foliar disease of soybean in the US Gulf South, causing significant economic impacts throughout the growing region. The sole causal agent of CLB was thought to be Cercospora kikuchii, but we now recognize that the disease is predominantly caused by Cercospora cf. flagellaris and Cercospora cf. sigesbeckiae in North America. However, a lack of information about the spatial population dynamics of the causal agents and the disease cycle, including the most important sources of inoculum, hinders the development of new and effective management tools. Species-specific detection/quantification assays are a first step towards addressing these epidemiological knowledge gaps. By quantifying pathogen presence both spatially and temporally, we can better elucidate the disease cycle and identify targets for developing precise disease management tools. A TaqMan quantitative polymerase chain reaction (qPCR) assay was developed for C. kikuchii prior to the discovery that C. cf. flagellaris and C. cf. sigesbeckiae are the principal causal agents, but we found the assay was not species-specific, detecting both C. kikuchii and C. cf. flagellaris. Therefore, new TaqMan qPCR assays were developed for C . cf. flagellaris, C. cf. sigesbeckiae, and C. kikuchii by leveraging comparative genomics. The assays were species-specific and had a detection limit of ~3-7 pg/µl DNA, which corresponded to approximately 100 conidia. PCR efficiencies were between 93-105% for genomic DNA and 83-110% for conidial samples, depending on the assay. These assays can be used for the quantification of CLB pathogens to ultimately guide effective CLB disease management.