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<p>To detect pathogenic fungi in conifer seed lots from different continents, we applied DNA metabarcoding targeting the fungal ITS2 region using the MiSeq platform and analyzed the sequences with the FungiSearch pipeline developed in a previous study. Analysis of a mock community comprising twelve fungal species showed that the method had a detection limit comparable to that of a species-specific PCR. All twelve species were detected with high repeatability. However, the method proved to be non-quantitative, as relative abundances were under- or overestimated depending on the fungal species. Analysis of seed batches showed that the number of biological replicates influenced fungal community diversity. Several pathogenic fungal species were detected: some were known to be associated with conifer seeds, whereas others were not. Pathogenic fungi emerging in Europe were also detected. Specific molecular tests (PCR or qPCR) were performed on DNA from different seed batches to target eight of these pathogens. All the pathogens detected by high-throughput sequencing were also detected by specific molecular tests, although detection rates varied slightly between the two methods. Based on these results, the high-throughput sequencing method validated in this study could be proposed as a screening test detecting fungal pathogens in conifer seeds. Positive detection should be confirmed with specific tests, particularly for fungi not previously reported in Europe, or with limited distribution.</p>

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Validation of a metabarcoding-based screening method for detecting fungal pathogens in conifer seeds using Illumina MiSeq and the FungiSearch pipeline

  • 期刊论文

  • 发表日期：

    2026年5月26日

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  • Anne Chandelier,
  • Sophie Schmitz,
  • Benjamin Dubois,
  • Gilles San Martin

摘要

To detect pathogenic fungi in conifer seed lots from different continents, we applied DNA metabarcoding targeting the fungal ITS2 region using the MiSeq platform and analyzed the sequences with the FungiSearch pipeline developed in a previous study. Analysis of a mock community comprising twelve fungal species showed that the method had a detection limit comparable to that of a species-specific PCR. All twelve species were detected with high repeatability. However, the method proved to be non-quantitative, as relative abundances were under- or overestimated depending on the fungal species. Analysis of seed batches showed that the number of biological replicates influenced fungal community diversity. Several pathogenic fungal species were detected: some were known to be associated with conifer seeds, whereas others were not. Pathogenic fungi emerging in Europe were also detected. Specific molecular tests (PCR or qPCR) were performed on DNA from different seed batches to target eight of these pathogens. All the pathogens detected by high-throughput sequencing were also detected by specific molecular tests, although detection rates varied slightly between the two methods. Based on these results, the high-throughput sequencing method validated in this study could be proposed as a screening test detecting fungal pathogens in conifer seeds. Positive detection should be confirmed with specific tests, particularly for fungi not previously reported in Europe, or with limited distribution.