New Insights into DNA Extraction and PCR for Tiny Fungi in the Laboulbenia Genus

Jenn Hoskins
12th June, 2024

New Insights into DNA Extraction and PCR for Tiny Fungi in the Laboulbenia Genus

The delicate micromanipulation process used for successful DNA extraction involves carefully removing, dissecting, and transferring individual thalli of the minute fungus Laboulbenia galeritae from its host into a PCR tube (a–h).

Image adapted from: Caenegem et al. / CC BY (Source)

Key Findings

  • Researchers at Ghent University developed a cost-effective DNA extraction method for Laboulbenia fungi
  • Proper specimen preservation in >95% ethanol significantly improves DNA extraction success
  • Adding bovine serum albumin (BSA) did not significantly enhance DNA extraction success, suggesting melanin is not a major inhibitor
The study of fungi within the order Laboulbeniales, particularly the genus Laboulbenia, has long been challenged by the tiny size of these organisms, their inability to grow in pure cultures, and the lack of effective and economical DNA extraction methods. Researchers at Ghent University have made significant strides in overcoming these hurdles, as detailed in their recent study[1]. Laboulbeniales are a group of fungi known for their unique parasitic relationships with arthropods. Historically, their classification has been controversial, with some biologists misidentifying them as worms or red algae[2]. However, molecular and developmental studies have since confirmed their place among the ascomycetes, a large group of fungi characterized by their spore-producing structures[2]. Despite these advances, the molecular study of Laboulbeniales has been hampered by difficulties in DNA extraction and PCR amplification, particularly due to the presence of melanin in their cells, which inhibits PCR. The team at Ghent University evaluated a standard single cell-based DNA extraction protocol, modifying it to reduce costs and resource consumption. By halving the recommended amount of reagents and adding bovine serum albumin (BSA) during the multiple displacement amplification step, they aimed to reverse the inhibitory effects of melanin. Out of 196 extractions, 111 were successful, demonstrating that reducing reagent amounts did not significantly affect the success rate of DNA extraction. This finding is crucial as it suggests that cost-effective methods can be employed without compromising the quality of DNA extraction. Interestingly, the addition of BSA did not significantly improve the probability of successful DNA extraction. This indicates that the amount of melanin present in the cells of Laboulbenia does not have a major inhibitory effect on PCR, contrary to previous assumptions. The study generated 277 sequences from five loci, although amplification of certain regions, such as the internal transcribed spacer (ITS) and mitochondrial small subunit rDNA, remained challenging. The ITS region, commonly used for analyzing fungal diversity, has been shown to introduce taxonomic biases during PCR amplification. Previous studies have highlighted that different ITS primers can preferentially amplify certain fungal groups, such as basidiomycetes or ascomycetes, leading to skewed results[3]. This underscores the need for careful primer selection and the potential development of Laboulbeniales-specific primers to avoid mismatches and contaminants. The success of DNA extraction was also influenced by specimen storage methods. Material preserved in >95% ethanol yielded higher success rates compared to those stored in 70% ethanol or dried material. This finding emphasizes the importance of proper specimen preservation for molecular studies. The advancements made by the Ghent University team represent a significant step forward in the molecular study of Laboulbeniales. By developing cost-effective and efficient DNA extraction methods, they have paved the way for more comprehensive studies of these fungi. The new insights gained from this research will not only benefit the study of Laboulbenia but also the broader field of Laboulbeniales, many of which remain unsequenced and understudied. Overall, the study highlights the importance of optimizing DNA extraction protocols and proper specimen preservation to overcome the challenges associated with studying these minute fungi. The development of specific primers for Laboulbeniales will further enhance the accuracy and reliability of molecular analyses, ultimately aiding in the understanding of their true diversity and evolutionary relationships.

GeneticsBiochemMycology

References

Main Study

1) New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales)

Published 11th June, 2024

https://doi.org/10.1186/s43008-024-00146-9

Related Studies

2) Laboulbeniomycetes: Evolution, natural history, and Thaxter's final word.

https://doi.org/10.1080/00275514.2020.1718442

3) ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases.

https://doi.org/10.1186/1471-2180-10-189


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