Macrophomina phaseolina is an important pathogen threatening groundnut production, with climate change-induced variations in temperature and precipitation resulting in considerable yield reductions. This research provides a comprehensive evaluation of the root rot prevalence and severity in the primary groundnut-cultivating areas of Tamil Nadu. Findings indicate an incidence range of 32.71% to 85.66%. Pathogenic variability testing identified GMP-3 as the most virulent strain. Morphological differences and sporulation patterns were observed and M. phaseolina isolates were confirmed through ITS 1 and ITS 4-sequencing, yielding Polymerase chain reaction amplicons of approximately 560 bp, which were submitted to Gene Bank. Additionally, a survey was conducted to collect various wild mushroom species from different regions in Tamil Nadu. Phenotypic and morphological characterization and molecular confirmation of the mushroom isolates were performed. The efficacy of these isolates against M. phaseolina was evaluated. The Tricholoma equestre (AWM-4) isolate demonstrated the highest inhibition in dual culture assays at 74.33%. Mushroom extracts prepared using various polar and nonpolar solvents were tested using the poisoned food technique to assess the inhibition percentage of M. phaseolina mycelial growth. Methanol extracts exhibited the greatest reduction in mycelial growth, with an inhibition rate of 75.56%. Gas Chromatography-Mass Spectrometry (GC-MS) analysis was performed to identify the compounds with active antifungal properties.
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