Single Nucleotide Polymorphisms in Colletotrichum Cereale: A Method for Detecting Unique Isolates of the Turfgrass Anthracnose Fungus.

Over the past decade, anthracnose disease outbreaks caused by Colletotrichum cereale have been increasing in frequency and becoming more destructive on golf course putting greens.  DNA fingerprinting has revealed host-specific populations and considerable diversity within the species, with isolates forming two distinct lineages.  Although these markers are able to identify wide-scale differences between populations, fine-scale multilocus markers containing greater signal are required to understand the trajectory of recent disease outbreaks caused by this pathogen.  In order to identify single nucleotide polymorphisms (SNPs) that might serve as the basis for developing high-resolution molecular markers for C. cereale, 28,699 Illumina sequenced restriction associated DNA (RAD) tags were generated from 54 isolates of Colletotrichum from grasses and cranberry.  The greatest number of polymorphic loci were identified from grass isolates, with on average, 1,042 alleles per isolate identified for C. cereale, with each sequence containing 1-6 SNPS.  This high degree of variation is ideal for developing fine-scale, sub-specific molecular markers and has been used to implement real-time PCR primers and probes to quickly and accurately identify C. cereale isolates belonging to each lineage.  Development of additional sub-specific markers to detect further differences between isolates is currently underway.  The creation of such markers will allow for culture independent assay methods and detection of unique C. cereale individuals through the use of real-time PCR; a fast, reproducible and reliable method that can be implemented by both diagnosticians and turfgrass pathologists.