Optimized protocol for culturing and extracting DNA from fungal isolates associated with brown spot needle blight in pine trees

by Temitope Ruth Folorunso, Gabriel Silva, Marilis E. Girón, Tess Lindow, Micah Persyn, Lori Eckhardt, Janna R. Willoughby

Effective culturing and DNA extraction protocols are essential for advancing research on fungal pathogens of brown spot needle blight (BSNB) that infect loblolly pine (Pinus taeda) and other Pinus species. We evaluated the performance of four widely used fungal media, including Sabouraud dextrose, malt extract, potato dextrose, and yeast extract peptone dextrose, in both solid (agar) and liquid (broth) formats, quantifying fungal growth through colony diameter and biomass accumulation over a three-week period. Sabouraud dextrose agar and broth consistently supported the most rapid and extensive growth in both formats, while potato dextrose underperformed across these metrics. To identify an optimal protocol for downstream molecular applications, we also compared four DNA extraction methods, three of which were modified variants of the CTAB (cetyl-trimethyl-ammonium bromide) chemistry as well as the Qiagen DNeasy kit following the yeast DNA extraction protocol. DNA yield, quantified by fluorometry, was highest for the high-salt CTAB polyvinylpyrrolidone (PVP) protocol and DNA purity (assessed by 260/280 absorbance ratio) was optimal for both PVP and Qiagen extractions. From these comparisons, we suggest that Sabouraud dextrose culturing combined with CTAB PVP extraction for use as a robust and accessible pipeline for generating high-quality fungal DNA.