Combination of a hierarchical and thermal shock process with a specific aqueous buffer: a safe, rapid and reliable DNA extraction method for plant endophytic fungi.

This study aimed to investigate the utilization of a buffer solution, containing Tris, HCl, EDTA, NaCl, and glycerol (THENG), in combination with a thermal shock process, as a novel DNA extraction method (THENG-tsp) for endophytic fungi. Three mg of fungal mycelium was added to 100 μL of THENG buffer and incubated in a water-bath (83 ± 2 °C) for 35 min. The vials were subsequently frozen at -20 °C for 15 min and then subjected to centrifugation (11,200 m/s2; 6 min). The resulting supernatant was used as a pure DNA source. In addition, the DNA of endophytic fungi was extracted using the cetyl trimethylammonium bromide (CTAB) method. DNA quality was evaluated based on light intensity and distance (in pixels) of bands observed on a 0.8% agarose gel after electrophoresis. The results indicate that the application of the THENG-tsp method improved the average band quality of all endophytic fungi by 7.86%. More precisely, bands corresponding to endophytic fungi belonging to the Basidiomycota and Ascomycota phyla exhibited an average increase in band quality of 13.50% and 8.57%, respectively, compared to the CTAB method. The application of the THENG-tsp method also resulted in improved band quality for Alternaria spp. and Fusarium spp., with average increases of 8.47% and 28.40%, respectively, compared to the CTAB method. Hence, the THENG-tsp method is considered appropriate for DNA extraction from endophytic fungi in laboratory settings due to its minimal requirement of initial fungal mycelium, time efficiency, and the absence of the use of potentially harmful chemical compounds. [ABSTRACT FROM AUTHOR]