Development and validation of real-time PCR for in-wood detection of <i>Ceratocystis ficicola</i> , the agent of canker, wood discoloration, and wilt in the common fig tree ( <i>Ficus carica</i> L.)

ABSTRACT Ceratocystis ficicola (CF) is a fungus causing a lethal disease of fig trees characterized by bark canker, wood discoloration, and wilt. First reported in Japan in the 1970s, the pathogen was recently detected in Greece and Italy, raising concern among Mediterranean countries where fig is an important crop. Consequently, the European Plant Protection Organization (EPPO) placed the pathogen on the A2 list of quarantine organisms. Monitoring efforts have been hindered by difficulties in pathogen isolation and the lack of reliable diagnostic tools. To address these limitations, we developed and validated a real-time PCR assay using the intercalating dyes EvaGreen and SYBR Green with primers targeting the ITS1 region of the ribosomal RNA operon. Amplification efficiency and selectivity were assessed using serial dilutions of CF genomic DNA spiked with various wood extracts. Efficiencies ranged from 95.7% to 102.6%, demonstrating no inhibition by host tissues. The EvaGreen assay consistently detected 5 fg of genomic DNA per reaction, with an operational limit of detection (LoD) of 10 fg (Ct 35–36), while the SYBR Green assay achieved an operational LoD of 3 fg. Cloning the CF ITS region into a plasmid vector indicated that these LoDs correspond to ~123 and ~37 target copies. The assay showed complete specificity against C. platani and 49 additional non-target fungi and full inclusivity for 8 CF isolates. Validation on 114 samples from artificially inoculated, naturally infected, and non-infected trees confirmed optimal diagnostic performance. This assay provides a sensitive and robust tool for detecting and monitoring CF in fig. IMPORTANCE Diagnostic assessments for pathogenic microbes are fundamental to medical and plant pathology, supporting curative and preventive diagnosis, and enabling detailed investigations of disease biology, epidemiology, and resistance. Ficus carica (fig) is a key fruit crop in the Mediterranean basin and a characteristic component of its rural landscapes, where it has been cultivated since the early Neolithic. Ceratocystis ficicola (CF) is a fungus causing lethal bark canker and wilt of fig in Japan since the 1970s and was recently detected in Greece and Italy, raising concern among Mediterranean National Plant Protection Organizations, including those of major non-EU producers. These developments prompted the European Plant Protection Organization (EPPO) to place the pathogen on the A2 list of quarantine organisms, thereby laying the groundwork for mandatory phytosanitary measures. However, effective monitoring has been hampered by difficulties in pathogen isolation and the absence of reliable diagnostic tools. The severe damage caused by CF, its rapid local and long-distance spread, and its poor eradicability highlight the urgent need for a robust integrated diagnostic framework in which real-time PCR plays a central role in disease surveillance. This study represents an initial step toward that goal.