Development of a Quantitative SYBR Green Real-Time PCR for <i>Trichoderma afroharzianum</i> , Causal Agent of Ear Rot of Maize

<i>Trichoderma</i> <i>afroharzianum</i>, the causal agent of Trichoderma ear rot, is an emerging pathogen of maize (<i>Zea mays</i> L.). It was recently reported as a maize pathogen in Germany, France, and in Italy. In 2023, nine seed lots from three farms in Northern Italy were tested for their phytosanitary conditions, revealing infection rates of up to 71% with <i>Trichoderma</i> spp. All seed lots showed symptoms of Trichoderma ear rot infection, and 26 out of 50 isolates were identified as <i>T. afroharzianum</i>. The study confirmed that <i>T. afroharzianum</i> infects maize seeds causing severe disease. Thirteen isolates from infected seeds were used to design species-specific primers on the translation elongation factor 1α gene and to develop a SYBR Green quantitative PCR to detect and quantify <i>T. afroharzianum</i> in maize seeds. The assay was validated following EPPO standard PM 7/98 guidelines, assessing analytical sensitivity, specificity, selectivity, repeatability, and reproducibility. The specificity of the method was validated using 19 <i>T. afroharzianum</i> strains and 16 nontarget species, including <i>Trichoderma</i> species belonging to the <i>T. harzianum</i> species complex. Only target DNA produced positive amplifications. Analytical sensitivity was tested using serial dilutions of <i>T. afroharzianum</i> DNA, revealing a detection limit of 50 fg, even in the presence of maize seed DNA. The assay enables specific and sensitive detection of target DNA in asymptomatic samples, providing a valuable tool for early target detection and quantification during seed certification.