Development and validation of a 28S rDNA-based TaqMan real-time PCR assay for sensitive detection of Rhizoctonia theobromae in cassava tissues

Abstract Cassava witches’ broom disease (CWBD) is an important disease that causes significant production losses. The disease was initially attributed to phytoplasmas; however, inconsistent detection has raised questions regarding their association with the disease. Recent studies have increasingly associated Rhizoctonia theobromae (syn. Ceratobasidium theobromae ) with CWBD, highlighting the need for a more reliable detection method to support phytosanitary, surveillance, and disease management strategies. This study developed and validated a TaqMan-based real-time PCR assay targeting a species-specific 101-bp region of the 28S rDNA of R. theobromae . Primers and probe were designed using Geneious Prime and evaluated for specificity using Primer-BLAST and in vitro assays. No cross-reactivity was detected with DNA from cassava-associated Fusarium, Colletotrichum , Cladosporium , Lasiodiplodia , and other fungi isolated from cassava tissues. Analytical sensitivity was evaluated using 10-fold serial dilutions of R. theobromae genomic DNA from 1 ng/µL to 0.001 fg/µL. The assay consistently detected 10 fg/µL DNA, establishing an analytical limit of detection. The standard curve showed an amplification efficiency of 99.77%, an R² of 0.987, and a slope of − 3.33. Application of the assay to symptomatic cassava plants demonstrated detection of R. theobromae DNA in leaf petiole/midrib, stem, and root tissues, with stem tissue showing the highest detection frequency (95.2%) at harvest stage. Target DNA was also detected in 5 of 33 asymptomatic leaf petiole/midrib samples (13.2%). These findings demonstrate that the developed real-time PCR assay enables sensitive and specific molecular detection of R. theobromae and has potential applications in CWBD surveillance, epidemiological studies, and phytosanitary diagnostics.