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

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.