Early and sensitive detection of Ceratocystis ficicola in Ficus carica: a new Taqman real-time PCR approach.

Ceratocystis ficicola is a soil-borne pathogen that infects the roots, the trunk and main stem of susceptible hosts, causing vascular wilt and tree death. It was first identified as the causal agent of Ceratocystis canker in fig (Ficus carica) in Japan and subsequently reported in Greece and Italy. Diagnostic procedures for C. ficicola are not yet fully standardized. In this study, specific primers and a TaqMan real-time PCR assay were designed and implemented to develop a rapid, sensitive molecular diagnostic tool for C. ficicola detection. Prior to assay validation, the identity of the fungal isolates was confirmed by evaluating key morphological traits, including the size and shape of ascomata and conidia, and by amplifying the internal transcribed spacer (ITS) region using primers ITS5 and ITS4. Assay validation was conducted on a panel of fungal isolates collected from healthy and diseased F. carica plants. Additional tests were performed on symptomatic, asymptomatic, and artificially inoculated plant and soil samples, demonstrating the assay’s ability to reliably detect the pathogen. The assay exhibited high sensitivity, detecting as little as 0.2 pg μL-1 of Ceratocystis DNA both in water and in plant DNA extracts. These findings indicate that the molecular method developed, utilizing specific primers and a TaqMan probe, offers a reliable, sensitive, and time-efficient tool for C. ficicola detection. Implementation of this assay will facilitate improved monitoring of the pathogen’s distribution, particularly in the Apulian region, and support timely management strategies to mitigate its spread.