A Reliable In Planta Inoculation and Antifungal Screening Protocol for Rhizoctonia solani-Induced Sheath Blight in...

Sheath blight, caused by Rhizoctonia solani, is a major fungal disease of rice that leads to significant yield losses globally. Conventional inoculation methods often fail to achieve consistent and uniform infection, limiting their applicability in antifungal screening studies. This protocol describes a reliable in planta inoculation method for R. solani using mature sclerotia placed at the internodal region of tillering-stage rice seedlings. The procedure includes step-by-step instructions for seed germination, seedling preparation, pathogen culture, artificial inoculation, and post-infection application of antifungal treatments, including botanical compounds such as Ocimum gratissimum essential oil and thymol. Lesion development is monitored and quantified over time, and data are analyzed statistically to evaluate treatment efficacy. The protocol is optimized for reproducibility, scalability, and compatibility with sustainable disease management approaches. It provides a robust platform for evaluating antifungal agents in a biologically relevant and controlled environment. Key features • Establishes a reliable in planta inoculation method for R. solani in rice, overcoming the common challenge of achieving consistent disease development. • Enables post-inoculation screening of botanicals for antifungal efficacy under realistic plant-pathogen interaction conditions. • Integrates sustainable research practices by detailing botanical extraction and their in planta assessment against R. solani infection.