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

Abstract

Quambalaria eucalypti is a fungal pathogen that causes leaf spot, shoot blight, and stem canker on Eucalyptus spp. Early diagnosis of the disease is difficult, although the symptoms are clear in its advanced phase. To enable a rapid and sensitive screening of asymptomatic or latently infected plant material for Q. eucalypti, a SYBR green-based real-time PCR assay targeting the partial histone-H3 region was developed. The assay demonstrated specificity for Q. eucalypti, not showing cross-reactivity with other Quambalaria species or the other eucalyptus fungal pathogens tested. 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. The robustness and efficacy of the assay was demonstrated by interlaboratory comparisons with similar results. This newly developed quantitative PCR assay can be used for more comprehensive epidemiological investigations, testing the plant material in known Q. eucalypti distribution areas for early management strategies, or collecting data for resistance breeding programs.