Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR.

Abstract

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = -0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = -0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = -0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.