Introduction: A new era of microbiome profiling in crops
Understanding the microbial communities living on wheat leaves is crucial for crop health and yield. Traditional short-read ribosomal barcodes often miss fine-scale diversity within dominant phyllosphere taxa. A recent approach leverages pangenome-informed, taxon-specific long-read amplicons to achieve species- and strand-level resolution in real agricultural settings. Here, we summarize how a field-based study on eight elite European winter wheat varieties demonstrates the power and practicality of this method across time points, canopy heights, and plant cultivars.
Study design: field sampling and mock communities
Eight winter wheat cultivars were sampled in two blocks across five timepoints during the growing season and at three leaf positions per plant. Leaves were collected under natural infection by Zymoseptoria tritici, with no fungicide applications. To validate the method, three mock communities were prepared, combining ten Pseudomonas strains and two Z. tritici isolates in varying DNA dilutions. This setup enabled a robust assessment of detection limits, primer performance, and profiling accuracy in both controlled and natural samples.
Pangenome-informed amplicon design: expanding resolution beyond 16S/ITS
The core innovation lies in designing 3-kb long amplicons based on pangenome analyses. For Pseudomonas, 19 representative genomes formed a pangenome from which 1059 core regions were identified; eight highly polymorphic amplicons were shortlisted for evaluation. For Z. tritici, a global pangenome provided two top amplicons on chromosomes 9 and 13. These locus-specific references were tested against mock cultures and field leaf samples to ensure broad representation of natural diversity while maintaining taxon specificity.
Primer design and validation
Candidate amplicons underwent multiple sequence alignment, consensus sequence construction, and Primer3-based primer design. We allowed limited degeneracy to maximize coverage across diverse strains. Amplicons were evaluated against thousands of genomes, with reads assigned by BLASTn and validated by Sanger sequencing where feasible. The final workflow included PCR optimization, including annealing temperature gradients and touchdown protocols, to ensure consistent amplification across unique leaf matrices and timepoints.
Methods: high-fidelity sequencing and analysis
Leaf samples were lyophilized, homogenized, and subjected to a refined DNA extraction protocol to enrich both bacterial and fungal DNA. Long-read sequencing libraries were prepared for two amplicon sizes (3 kb and 1.5 kb) and sequenced on PacBio Sequel II with HiFi accuracy (>99%). After base calling, reads were demultiplexed by barcodes, and primer sequences were trimmed. Amplicons were classified with BLAST against reference databases and filtered to retain high-quality, locus-specific ASVs. Diversity metrics were computed from haploid-style VCF-derived alleles to quantify nucleotide diversity across amplicons.
Results: resolution, detection limits, and ecological insights
The Pseudomonas-focused amplicons (rpoD and ABC transporter locus) detected far more ASVs than full-length 16S from the same samples, enabling finer species- and subspecies-level discrimination. In field samples, rpoD and transporter amplicons resolved diverse Pseudomonas groups, including several subspecies of P. chlororaphis, with distinct abundance patterns across time and canopy height (bottom vs. top leaves). Z. tritici amplicons on chromosomes 9 and 13 captured substantial strain-level diversity that surpassed what ITS could reveal, highlighting the dynamic fungal population during an epidemic season.
Across timepoints, we observed consistent patterns: some Pseudomonas subspecies thrived in July on lower leaves, while others dominated earlier in the season on upper leaves. This reveals temporal and spatial structuring of plant-associated microbiomes previously masked by less-resolved markers. The methodology also demonstrated robust performance in highly multiplexed experiments, achieving near-10,000 reactions multiplexed in a single PacBio run with high-quality, long reads.
Implications and future directions
Taxon-specific, long-read amplicons crafted from pangenomes offer a scalable route to decipher microbial ecology in crops and other complex ecosystems. By providing species- and strain-level resolution, this approach strengthens our ability to link microbial dynamics with plant health, disease suppression, and biocontrol potential. Beyond Pseudomonas and Z. tritici, similar pipelines can be extended to Rhizobia, Streptomyces, Aspergillus fumigatus, and other ecologically relevant groups, enabling broad comparative studies and targeted manipulation of microbiomes for sustainable agriculture.
Conclusion: a practical, scalable solution for high-resolution microbiome profiling
Long-read, pangenome-informed amplicon design delivers precise, taxon-resolved insights that surpass traditional ribosomal markers. In the wheat phyllosphere, this method unpacks complex, dynamic communities and identifies candidate biocontrol taxa at subspecies and strain levels—promising a new era of precision microbiome management for crop health and resilience.
