ARS | Publication request: Culture-independent, real-time PCR typing of Colletotrichum cereale specimens from modern and historical collections using the Apn2 marker

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 1, 2011
Publication Date: June 15, 2011
Citation: Beirn, L., Clarke, B., Crouch, J. 2011. Culture-independent, real-time PCR typing of Colletotrichum cereale specimens from modern and historical collections using the Apn2 marker [abstract]. American Phytopathological Society, August 6-10, 2011, Honolulu, HI. paper no. 101:S14.

Technical Abstract: Anthracnose, caused by the fungus Colletotrichum cereale, is a destructive disease of cool-season turfgrasses. The fungus is also a common inhabitant of cereals, field, forage and prairie grasses. Molecular analysis of C. cereale has revealed considerable diversity within the species, with isolates forming two primary lineages, designated clade A and clade B. Research of this fungus is limited by the need to establish pure cultures of C. cereale, a time-consuming process that requires several sub-culturing steps and often fails due to the presence of faster growing organisms. To test whether C. cereale could be detected directly from DNA extracted from infected plant tissue, two real-time PCR probes specific for clades A and B were developed using the single-copy Apn2 (DNA lyase) gene as a template for marker design. Over 530 cultured isolates of C. cereale from turfgrass, prairie, and wheat hosts were screened using the probes, along with 75 field samples collected from a diseased turfgrass putting green, 50 asymptomatic wheat plants, and 106 herbarium specimens on various host substrates. The probes were 100% accurate for C. cereale detection from cultured samples, and for the discrimination of known clade A and clade B isolates (avg. cycle threshold (CT): A=28.15; B= 26.82). C. cereale was successfully detected in a clade-specific manner from diseased field samples and 68-103 year-old herbarium specimens (avg. CT =24.88 and 34.75, respectively). Using this assay, type specimens of the fungus collected in 1908 were identified as C. cereale, validating previous morphology-based assessments. These probes will be useful for culture-independent, high-throughput molecular analysis of the distribution and incidence of C. cereale populations.