Development of a qPCR assay for species-specific detection of the tar spot pathogen Phyllachora maydis

Phyllachora maydis is a fungal plant pathogen that causes tar spot of corn (Zea mays) in North and South America causing devastating yield losses under favorable conditions. Though the causal agent is relatively easy to diagnose via macroscopic and microscopic observations, other diseases and conditions such as insect frass have been mistaken for tar spot of corn. Furthermore, conidia and ascospores in isolation can be difficult to visually distinguish from other fungi, and the development of signs and symptoms of the disease may not be observed until 12 to 20 days after infection. Therefore, we developed a TaqMan quantitative polymerase chain reaction (qPCR) assay for the detection and quantification of this pathogen to be used for diagnostics and airborne spore quantification. The assay was designed to the internal transcribed spacer (ITS) region of P. maydis. The specificity of the assay was confirmed and tested against various non-target Phyllachora species, corn pathogens, endophytes, and P. maydis samples from several states in the Midwest and from Mexico. The detection limit of this assay was determined as 100 femtograms (fg) of genomic P. maydis DNA. To demonstrate the transferability of this technology, the assay was tested in different labs using various qPCR thermal cyclers. This assay can be used in downstream research involving latency period, disease prediction, and diagnostics.