Pioneering Nit Gene Exploitation to Develop Molecular Diagnostic Assay for Rapid Detection of Cotton Root Rot Incitant, Macrophomina phaseolina (Tassi) Goid, in Field Soil

Cotton root rot caused by Macrophomina phaseolina pose a significant threat to cotton production, leading to substantial yield and quality losses. Early and accurate diagnosis of this pathogen in soil is crucial for effective disease management. This study presents a pioneering investigation into the utilization of the nit gene encoding nitrilase for the development of a molecular diagnostic assay aimed at the rapid detection of M. phaseolina in field soils. The methodology involved the design and validation of primers targeting the Nit gene sequence, followed by the optimization of PCR conditions for efficient amplification. Leveraging state-of-the-art molecular techniques, the assay offers a novel protocol to accurately identify the presence of M. phaseolina in soil with high sensitivity and specificity. The specificity of the designed primers was confirmed through PCR amplification using DNA from M. phaseolina and other related fungi. Sensitivity tests demonstrated that the PCR assay reliably detected M. phaseolina DNA at concentrations as low as 1 ng. Furthermore, the performance of the diagnostic assay was rigorously evaluated using field soil samples with a known status of M. phaseolina infection, demonstrating its reliability and efficacy in real-world scenarios. This study introduces a novel molecular marker for the detection of M. phaseolina and offers a rapid and efficient means for screening M. phaseolina in large soil samples with minimal time and manpower.