Development of a multipurpose diagnostic marker using PCR, real-time PCR, and LAMP assays for rapid detection of Rhizoctonia solani in rice plants and soil.

Rhizoctonia solani AG1-IA is a major soil-borne pathogen responsible for sheath blight in rice, causing significant yield losses globally. Early, rapid, and accurate detection is essential for effective disease management, as traditional methods are time-consuming and less sensitive. In this study, we developed and validated a highly specific molecular marker, BrD3 targeting the bromodomain-containing Major Facilitator Superfamily (MFS) gene. This marker was successfully employed in PCR, quantitative PCR (qPCR), and Loop-mediated Isothermal Amplification (LAMP) assays for the detection of the pathogen in infected rice plants and soil samples. The PCR assay detected as low as 100 pg of DNA, while the qPCR assay demonstrated higher sensitivity, detecting 10 pg of DNA with high efficiency and accuracy (R2 > 0.99). Further, marker was utilized for the resistance evaluation against sheath blight disease of rice where highest copy no. of target gene was detected in susceptible rice variety Pusa Basmati 1121, and minimum copy no. was detected in resistant variety Tetep under inoculated conditions. The LAMP assay, optimized at 63°C with Hydroxy Naphthalol Blue (HNB) dye, provided rapid visual detection with a sensitivity of 1 ng DNA and no cross-reactivity with other rice pathogens. All three assays were validated in infected plants across multiple rice genotypes and soil samples of rice-wheat cropping system. The integrated diagnostic approach presented here offers a robust, specific, and field-deployable system for early detection and soil population monitoring of R. solani (AG1-IA) facilitating timely disease management.