Development of a Sheath Inoculation Protocol to Screen Rice Varieties for Resistance to Cercospora janseana.

Cercospora janseana (Racib.) O. Const. is a re-emerging fungal pathogen that causes Cercospora net blotch on rice. Previous research on resistance to C. janseana has primarily focused on foliar symptoms. Subsequently, sheath infection remains poorly characterized which hinders disease management efforts. This study developed and validated a reproducible sheath inoculation protocol under controlled conditions. Three inoculation methods (agar disc, spray, and drop) were evaluated with and without mechanical wounding. Lesions only formed with inoculation methods using wounding and the agar disc method produced the most consistent and uniform symptom development. Time-course analysis in the susceptible variety Cheniere revealed earlier lesion onset, more rapid expansion, and lower variability in the agar disc method compared to spray, confirming its suitability for phenotypic screening. The optimized protocol was applied across five independent trials involving four rice varieties. DG263L consistently exhibited minimal lesion development, confirming its resistance, while Cheniere showed extensive lesion growth, indicating high susceptibility. PVL03 and LaGrue displayed moderately susceptible reactions, with PVL03 developing significantly higher lesion lengths and AUDPC values than LaGrue in one-month-old plants. Although lesion onset was delayed in 45-day-old plants, disease progressed more rapidly once established. AUDPC analysis corroborated these trends, further distinguishing varietal responses. The protocol effectively discerned resistant, intermediate, and susceptible phenotypes, supporting its use in resistance screening. To our knowledge, this is the first controlled sheath inoculation method developed for Cercospora net blotch, offering a standardized approach for evaluating sheath-specific resistance and advancing the characterization of the C. janseana-rice pathosystem.