Development of a Real-Time PCR Assay for Early Detection of the Eucalyptus Pathogen Quambalaria eucalypti

Quambalaria eucalypti is a fungal pathogen that causes leaf spot, shoot blight, and stem canker disease in Eucalyptus spp. Although the advanced stages of the disease show clear symptoms, diagnosis of the early stages can be challenging. To enable fast and sensitive screening of asymptomatic or latent infected plant material for Q. eucalypti, a specific SYBR green-based real-time PCR assay targeting the partial histone-H3 region was developed. The assay was demonstrated to be specific for Q. eucalypti and did not give cross-reactivity with any of the other Quambalaria species examined or other eucalyptus pathogens. The primers developed in this study ensured high analytical sensitivity, allowing the detection of Q. eucalypti DNA concentrations as low as 10 fg DNA from asymptomatic plants. Comparable results were obtained in interlaboratory testing, demonstrating the assay's robustness and effectiveness in other laboratories. This newly developed quantitative PCR assay can be used for more comprehensive epidemiological investigations, testing plant material in known Q. eucalypti distribution areas for early management strategies, or collecting data for resistance breeding programs.