A quantitative PCR-based assay for the species-specific detection of Caliciopsis pinea, causal agent of caliciopsis canker disease of Pinus strobus

Caliciopsis pinea is the ascomycete plant pathogen that causes caliciopsis canker disease on North American Pinus strobus (eastern white pine). Infections result in downgrading of lumber due to canker formation and overall loss of vigor in P. strobus, which is a critical cover species throughout its native range. C. pinea is challenging to distinguish from closely related species (some of which can coinfect P. strobus) based on macroscopic and microscopic observations alone. C. pinea and other Caliciopsis species that infect conifers all have sign development that is indistinguishable to the naked eye and often indistinguishable using microscopy and exhaustive measurement. Additionally, symptoms may develop on a host without fruiting bodies, making molecular techniques necessary for detection and diagnosis. Improved sensitivity and specificity of diagnostic and detection tools are critical for the early identification of and response to C. pinea infections. Therefore, this study aimed to develop a quantitative polymerase chain reaction (qPCR) assay for the detection, diagnosis, and quantification of C. pinea from infected plant material. This assay targets the internal transcribed spacer (ITS) region of C. pinea, and specificity was confirmed on a panel of 51 target and nontarget isolates from throughout the P. strobus native range. The limit of detection for the assay was determined to be 10 fg of C. pinea DNA. The technology was transferred out to be tested in another lab, as well as another thermocycling platform to ensure reproducibility of results. This study reports the most sensitive and specific assay for C. pinea detection for our current understanding of species diversity within the genus.