Darwin Magsino Landicho

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.

Cassava witches’ broom disease (CWBD) is one of the most devastating diseases of cassava (Manihot esculenta Crantz), and it threatens global production of the crop. In 2017, a phytoplasma, Candidatus Phytoplasma luffae (Ca. P. luffae), was reported in the Philippines, and it has been considered as the causal agent, despite unknown etiology and transmission of CWBD. In this study, the nationwide occurrence of CWBD was assessed, and detection of CWBD’s pathogen was attempted using polymerase chain reaction (PCR) and next-generation sequencing (NGS) techniques. The results showed that CWBD has spread and become severe, exhibiting symptoms such as small leaf proliferation, shortened internodes, and vascular necrosis. PCR analysis revealed a low phytoplasma detection rate, possibly due to low titer, uneven distribution, or absence in the CWBD-symptomatic cassava. In addition, NGS techniques confirm the PCR results, revealing the absence or extremely low phytoplasma read counts, but a surprisingly high abundance of fastidious and xylem-limited fungus, Ceratobasidium sp. in CWBD-symptomatic plants. These findings cast doubt over the involvement of phytoplasma in CWBD and instead highlight the potential association of Ceratobasidium sp., strongly supporting the recent findings in mainland Southeast Asia. Further investigations are needed to verify the etiology of CWBD and identify infection mechanisms of Ceratobasidium sp. to develop effective diagnostic and control methods for disease management.