Development and validation of conventional and TaqMan real-time PCR for the detection of Trichoderma afroharzianum causing corn ear rot

The emergence of a new corn ear rot was recently described in Germany and has since received increased attention following detections in several European and Asian countries. All reports consistently identify Trichoderma afroharzianum as the causal agent. The potential impact on maize production, combined with the risk of misdiagnosis as other ear rot diseases, highlights the need for a rapid and accurate diagnostic method for the reliable detection of T. afroharzianum. In this study, we developed a PCR-based diagnostic tool including an endpoint PCR and a real-time TaqMan assay targeting the TEF1α and RPB2 loci, respectively. Both assays were validated using a broad panel of Trichoderma isolates and other fungal species obtained from maize plants, additional host species from diverse geographic origins, and soils collected from German maize fields. All performance criteria required to demonstrate assay reliability were met. The two PCR assays demonstrated higher analytical specificity and inclusivity, albeit were unable to distinguish between pathogenic and non-pathogenic T. afroharzianum isolates. They showed high accuracy and sensitivity, with limits of detection of 1 pg µL⁻¹ for the endpoint PCR and 1 fg µL⁻¹ for the real-time PCR. The real-time assay exhibited amplification efficiencies of 105,5% (R² = 0.977) for pure cultures and 116% (R² = 0.997) for infected maize kernels. Furthermore, both assays reliably detected T. afroharzianum in asymptomatic maize cobs following artificial inoculation with pathogenic strains. These methods therefore enable early and reliable detection of T. afroharzianum ear rot and allow its discrimination from other maize ear rot diseases. This capability is essential for the development of targeted management strategies to address this emerging threat to maize production.