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Profiling grapevine trunk pathogens in planta: a case for community-targeted DNA metabarcoding.

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Custom DNA metabarcoding primers (GTAA) accurately detect grapevine trunk pathogens, outperforming universal primers in sensitivity and quantitative profiling. Primer design is crucial for reliable results in fungal community analysis.

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Area of Science:

  • Molecular Ecology
  • Plant Pathology
  • Mycology

Background:

  • DNA metabarcoding is valuable for identifying multiple pathogens in complex diseases like grapevine trunk diseases.
  • Profiling fungal communities in grapevine wood is challenging due to diverse co-infecting species.
  • Existing universal primers may not accurately represent pathogen abundance in complex samples.

Purpose of the Study:

  • To design and evaluate novel metabarcoding primers (GTAA) for grapevine trunk-associated ascomycete fungi.
  • To compare the performance of GTAA primers against widely used universal primers for fungal community profiling.
  • To assess the accuracy and sensitivity of DNA metabarcoding for grapevine trunk disease diagnostics.

Main Methods:

  • In silico simulations and high-throughput amplicon sequencing were employed.
  • Testing involved mock communities, time-course inoculation experiments, and field-collected diseased samples.
  • GTAA primers were designed using the ribosomal internal transcribed spacer region.

Main Results:

  • GTAA primers demonstrated superior affinity and sensitivity compared to universal primers.
  • GTAA accurately profiled fungal genera (Phaeomoniella, Phaeoacremonium, Eutypa) in mock communities and field samples.
  • Universal primers led to over- or under-estimation of key trunk pathogen abundances.

Conclusions:

  • DNA metabarcoding provides accurate qualitative and quantitative data for grapevine trunk disease studies.
  • Customized primer design and rigorous testing are essential for valid DNA metabarcoding results.
  • The GTAA primer set offers an improved tool for studying grapevine trunk fungal pathogens.