Multiplex real-time PCR detection and differentiation of colletotrichum species infecting soybean

Colletotrichum species are fungal plant pathogens of worldwide significance. Colletotrichum species were isolated from soybean with anthracnose symptoms in five states in the United States from 2009 to 2013. Among 240 isolates collected, four Colletotrichum species were initially identified by morphological and sequence analysis, including C. chlorophyti, C. incanum, C. truncatum, and Colletotrichum sp. (henceforth Glomerella glycines, the name of its sexual state). To increase diagnostic efficiency and accuracy, real-time multiplex PCR assays based on a double-stranded DNA-binding dye coupled with dissociation curve analysis were designed, using a region of the cytochrome c oxidase subunit 1 (cox1) gene to discern these four Colletotrichum species. Two sets of duplex, real-time PCR assays were established and species differentiation was based upon amplicon melting point temperatures (Tm) in the dissociation curve analysis. The Set 1 duplex assay distinguished C. chlorophyti and G. glycines, and the Set 2 duplex assay distinguished C. incanum and C. truncatum. Successful detection was achieved with as little as 1 pg DNA. The assays were especially useful for differentiating C. chlorophyti, C. incanum, and C. truncatum, which have similar morphological features. Colletotrichum gloeosporioides, another pathogen associated with soybean anthracnose, was not resolved from G. glycines by the melting curve analysis. The two duplex real-time PCR assays were used to screen more than 200 purified Colletotrichum isolates, showing that they were rapid and effective methods to detect and differentiate Colletotrichum species infecting soybean.