Advancing Fungal Genetics Through an Integrated gDNA Extraction Approach Across Soil, Rhizosphere, and Root Compartments

Efficient and reliable extraction of high-quality genomic DNA (gDNA) from complex environmental matrices remains a critical challenge in fungal ecology and plant–microbe interaction studies. Here, we present an optimized and integrated methodology for gDNA extraction from soil, rhizosphere, and root endosphere samples, with particular emphasis on differentiating endophytic from epiphytic fungal communities. Using maize grown in contrasting substrates (RPM soil and sand) and inoculated with Trichoderma variants, we evaluated surface sterilization strategies, culture-based and non-culture-based DNA extraction methods, and downstream molecular validation using ITS PCR and sequencing. Nutrient-rich RPM soil consistently yielded higher DNA concentrations compared to sand, reflecting greater microbial abundance. Optimized sterilization protocols reduced epiphytic contamination while preserving endophytic signals, although complete surface sterility remained challenging. Molecular analyses confirmed successful amplification and identification of fungal DNA, demonstrating the robustness of the approach for monitoring fungal biostimulants. Overall, this integrated protocol improves DNA yield, purity, and reproducibility across multiple plant–soil compartments and provides a practical framework for fungal community analysis in complex agroecosystems.