High-sensitivity ITS real-time PCR assays for detection of Ceratocystis lukuohia and Ceratocystis huliohia in soil and air samples

Rapid Ohia Death (ROD) is caused by two pathogens, Ceratocystis lukuohia and Ceratocystis huliohia. The established species-specific real-time PCR assays targeting the single-copy cerato-platanin gene require DNA between 2 and 16 spores per reaction for consistent detection, which is suitable for analysis of infected plant tissue but not sensitive enough to consistently detect low spore-loads in environmental samples. Here we present two redesigned qPCR assays targeting the first Internal Transcribed Spacer (ITS) region of the multi-copy ribosomal DNA operon from the respective species, both capable of consistent detection of the pathogen at concentrations as low as 1 fg per reaction, less than the size of the haploid genome. Due to this increased sensitivity, these ITS qPCR assays are superior for analysis of DNA extracted from material collected in airborne particle samplers and from soil. Here, we demonstrate the utility of these qPCR assays for the characterization of windblown and soilborne dispersal of the pathogens, which is critical for the development of management practices that mitigate disease spread.