Quantification of dollar spot inoculum (Clarireedia spp.) in bentgrass clippings using droplet digital polymerase chain reaction (ddPCR)

Authors: GHIMIRE, KRISHNA - University Of Nebraska; KIM, YONGHYUN - Orise Fellow; Barnaby, Jinyoung; Warnke, Scott

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2025
Publication Date: 6/17/2025
Citation: Ghimire, K., Kim, Y., Barnaby, J.Y., Warnke, S.E. 2025. Quantification of dollar spot inoculum (Clarireedia spp.) in bentgrass clippings using droplet digital polymerase chain reaction (ddPCR). Crop Science. https://doi.org/10.1002/csc2.70121.
DOI: https://doi.org/10.1002/csc2.70121

Interpretive Summary: Dollar spot disease of turfgrass is one of the most widespread and economically significant turfgrass diseases. Understanding how the dollar spot fungus interacts with resistant and susceptible cultivars is crucial for accurately assessing the mechanisms of dollar spot resistance. To achieve this, ARS scientists developed a precise and sensitive method for quantifying the dollar spot fungus in clipping samples. The creeping bentgrass cultivars “007XL”, “Penncross”, “Oakly”, and the colonial cultivar “Musket”, known to differ in dollar spot susceptibility, were monitored at the National Turf Evaluation Program (NTEP) Fairway trial in College Park, MD. The developed assay was highly effective for detecting dollar spot in clipping samples. A positive correlation between the dollar spot fungus levels and the severity of disease symptoms in the field was observed. Furthermore, the results revealed that resistant cultivars maintain relatively low levels of the dollar spot fungus early in the season, whereas the susceptible cultivar showed high dollar spot fungus levels and severe disease symptoms. Later in the season, resistant cultivars tolerated higher dollar spot fungus levels before disease symptoms became visible. The susceptible cultivar exhibited two distinct epidemics, with corresponding peaks in dollar spot fungus and disease symptoms. In contrast, the resistant cultivars effectively avoided the first epidemic cycle, presumably by inhibiting growth of the dollar spot fungus. These results will facilitate breeding resistant cultivars and may improve the management of this important turfgrass disease.

Technical Abstract: Dollar spot, caused by the fungal pathogen Clarireedia jacksonii, is one of the most widespread and economically significant turfgrass diseases. Understanding how Clarireedia interacts with resistant and susceptible cultivars is crucial for accurately assessing the mechanisms of dollar spot resistance. To achieve this, a precise and sensitive method for quantifying Clarireedia in clipping samples was developed. Every week throughout the growing season Droplet Digital PCR (ddPCR) of clipping sample extracted DNA was used to quantify Clarireedia levels compared to the seasonal disease progression of three creeping and one colonial bentgrass cultivars. The creeping bentgrass cultivars, “007XL”, “Penncross”, “Oakly”, and the colonial cultivar “Musket” known to differ in dollar spot susceptibility were monitored at the National Turf Evaluation Program (NTEP) Fairway trial at the University of Maryland, College Park. The results demonstrate that the ddPCR assay along with our newly designed primers and probe were highly effective for detecting and quantifying Clarireedia DNA at concentrations in clipping samples as low as 1 × 10'¹4 g. A positive correlation between Clarireedia DNA copy number and the severity of disease symptoms in the field was observed. Furthermore, the results revealed that resistant cultivars maintain relatively low levels of Clarireedia early in the season, whereas the susceptible cultivar showed high Clarireedia levels and severe disease symptoms. Later in the season, resistant cultivars tolerated higher Clarireedia levels before disease symptoms became visible. The susceptible cultivar exhibited two distinct epidemics, with corresponding peaks in Clarireedia DNA copies and disease symptoms. In contrast, the resistant cultivars effectively avoided the first epidemic cycle, presumably by inhibiting Clarireedia growth.