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

Summary

Scientists developed an optimized method for growing brown spot needle blight fungi from infected pine needles and extracting their DNA for research. They tested four different growth media and four DNA extraction techniques to find the best combination. Sabouraud dextrose medium combined with a CTAB-based extraction method containing polyvinylpyrrolidone worked best, producing high-quality DNA suitable for advanced genetic studies. This standardized approach will help researchers better understand this important forest disease.

Background

Brown spot needle blight (BSNB) is a fungal disease affecting at least 53 Pinus species worldwide, with increasing concern due to its occurrence in loblolly pine, which supports a large timber industry. The primary causal agent is Lecanosticta acicola, though other pathogenic fungi including endophytes are involved in this complex disease system. Effective culturing and DNA extraction protocols are essential for advancing molecular research on BSNB pathogens.

Objective

To evaluate the performance of four widely used fungal culture media and four DNA extraction methods to identify optimal protocols for culturing BSNB-associated fungi and extracting high-quality DNA for downstream molecular applications including long-read sequencing.

Results

Sabouraud dextrose agar and broth consistently supported the most rapid and extensive fungal growth, while potato dextrose underperformed across metrics. The 3% CTAB + polyvinylpyrrolidone (PVP) protocol produced the highest DNA yield (58.5 ± 4.8 ng/µL), while both 3% CTAB + PVP and Qiagen extractions achieved optimal purity ratios near 1.8.

Conclusion

Sabouraud dextrose culturing combined with 3% CTAB + PVP extraction provides a robust and accessible pipeline for generating high-quality fungal DNA from BSNB-associated fungi. This optimized protocol addresses a critical bottleneck in fungal genomics workflows and provides a practical template for adaptation to other fungal pathogen systems.
  • Published in:PLoS ONE,
  • Study Type:Comparative Experimental Study,
  • Source: 10.1371/journal.pone.0337218, PMID: 41259340
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