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Location: Soybean/maize Germplasm, Pathology, and Genetics Research

Title: Multiplex real-time PCR detection and differentiation of Colletotrichum species infecting soybean

item YANG, HUI-CHING - University Of Illinois
item Haudenshield, James
item Hartman, Glen

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2015
Publication Date: 11/1/2015
Citation: Yang, H., Haudenshield, J.S., Hartman, G.L. 2015. Multiplex real-time PCR detection and differentiation of Colletotrichum species infecting soybean. Plant Disease. 99:1559-1568.

Interpretive Summary: Fungi belonging to the genus Colletotrichum cause anthracnose on soybean worldwide with yield losses ranging from 16% to 100% in Brazil, India, Thailand, and southern areas in the United States. The pathogens can infect plants at any growth stage, although symptoms may be more apparent when the plants reach maturity, or under humid and warm conditions. This study developed molecular assays (dsDNA dye-binding based real-time PCR) to efficiently detect and differentiate pure isolates of Colletotrichum species infecting soybean. To our knowledge, this is the first report to utilize this method for identification and differentiation of phytopathogenic fungi at a species level. The current study showed the potential of using this method, which allowed for multiplex detection of fungal species causing anthracnose. The method can serve as a useful tool for disease diagnosis and investigation of soybean anthracnose and potentially other pathogens. This research will be useful for soybean pathologists, other crop pathologists, and diagnosticians that may want to use this assay for diagnosing species causing anthracnose.

Technical Abstract: Colletotrichum species are fungal plant pathogens of worldwide significance. We isolated Colletotrichum species from soybean [Glycine max (L.) Merr.] with anthracnose symptoms in the U.S. states of Alabama, Arkansas, Illinois, Mississippi, and North Dakota from 2009 to 2013. Thirty-five strains from 240 isolates were selected and identified by morphological characteristics and sequence analyses. Among them, four Colletotrichum species were obtained, including C. chlorophyti, C. incanum, C. truncatum, and Colletotrichum sp. (referred to within as Glomerella glycines, its sexual stage name). To increase diagnostic efficiency and accuracy, real-time multiplex PCR assays based on a double-stranded DNA-binding dye 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 the differentiation was based upon amplicon melting point temperatures 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 two duplex real-time PCR assays were used to identify more than 200 isolates in a Colletotrichum species collection showing that it was a rapid and effective method to detect Colletotrichum species infecting soybean.