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GenomicsPhytoFrontiers™
Peer-reviewed journal article

High Quality, Long-Read Genome Assemblies of Two Virulent Field Isolates of Pyricularia oryzae from Portugal

Pedro RosaiD, João Bilro, Ricardo S. RamiroiD, Cristina AzevedoiD

Abstract

The fungal pathogen Pyricularia oryzae is notorious for causing blast disease in various important cereal crops, including wheat, rice, millet, and oat. Whole-genome-informed data on this pathogen are necessary to better understand the host adaptability of the fungus, including identifying key determinants of infection to enable more precise disease control. Here, we report highly contiguous genome sequences (using long-read PacBio technology) of two isolates from rice paddies in Portugal, M22.7 and T22.2, which exhibit distinctly aggressive symptoms in rice. Both mitochondrial and nuclear sequences were characterised in this study. The resulting nuclear genomes have assembly lengths of 46.4 Mb for M22.7 (198x coverage) and 46.3 Mb for T22.2 (163x coverage), with near-complete BUSCO completeness (98.8%) and a 0% contamination score (EukCC). Phenotypic analyses on infected rice leaves show that both isolates are infectious and virulent in two different rice cultivars. Most notably, M22.7 lacks the avirulence effector genes Avr-Pii, Avr-Pia and Avr-Pik, which are present in T22.2. This announcement represents the first genome resource for natural isolates of P. oryzae from Portugal in over 30 years, filling an important data gap from a major European rice-producing country that produces locally adapted rice varieties under specific agro-environmental conditions (near the Atlantic coast).

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In-depth summary

✦ Multi-agent verified full-text synthesis

Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 2 (1 Oct 2026) · CC BY 4.0

Two Portuguese rice-blast fungus isolates were assembled from PacBio HiFi reads into near-complete genomes; one isolate lacks Avr-Pii, Avr-Pia and Avr-Pik yet remains virulent on two rice cultivars.

Only two Portuguese P. oryzae genomes were publicly available, both from isolates collected in the early 1990s, and none came from long-read sequencing. These assemblies add recent Portuguese isolates, which the authors say can inform the national rice breeding programme.

Key results

  1. ShownBoth isolates were assembled into highly contiguous, near-complete nuclear genomes with no detected contamination.

    M22.7: 46.4 Mb, 26 contigs, N50 4.73 Mb, 198x coverage. T22.2: 46.3 Mb, 15 contigs, N50 6.52 Mb, 163x coverage. Both: BUSCO completeness 98.8% (sordariomycetes_odb12), EukCC completeness 100%, contamination 0%. Each isolate has one complete 34866 bp mitochondrial genome with 44 genes. · Abstract; Genome Announcement

  2. ShownRead-mapping and reference comparisons indicate accurate assemblies, with T22.2 slightly cleaner than M22.7.

    Inspector QV 54.6 for M22.7 (one 85 bp collapse in ctg000080) vs QV 61.9 and no structural errors for T22.2. QUAST against the 70-15 reference: about 0.12% of assembled bases misassembled for both isolates. · Genome Announcement

  3. ShownIsolate M22.7 has no copies of the Avr-Pii, Avr-Pia or Avr-Pik effector genes, whereas T22.2 carries all three (Avr-PikA and Avr-PikD alleles included); even so, the two isolates infect both tested cultivars to a similar degree.

    Absence of Avr-Pik in M22.7 was confirmed by PCR. Detached-leaf lesion areas at 7 days post-infection in cv. Presto (n = 15 and 16 in two experiments) and cv. Bomba (n = 32 and 30) showed both isolates were infectious and virulent. · Abstract; Genome Announcement; Figure legends

  4. ShownAnnotation gives similar gene and repeat content in both isolates; M22.7 has Helitron insertions that T22.2 lacks and slightly fewer predicted effectors.

    Gene models: 13,065 (T22.2) vs 12,731 (M22.7) vs 13,184 for 70-15. Secreted proteins: 1765 vs 1688. Predicted effectors (apoplastic/cytoplasmic): 318/346 vs 309/322. Repeats: 19.1% in M22.7 vs 18.4% in T22.2; 24 Helitron insertions in M22.7, none in T22.2. · Genome Announcement; Figure legends

  5. SuggestedPhylogenetic placement and structural comparisons put both isolates in a rice-infecting clade; M22.7 differs structurally more than T22.2 and sporulates less in vitro.

    The two isolates form a fully supported branch sharing an ancestor with Portuguese isolate PR0009 (UFBoot > 94% across the tree). Dotplots against 70-15, FR13 and Guy11 show more inversions and transpositions in M22.7. M22.7 shows consistently reduced conidiation in vitro. · Genome Announcement

Methods, data and reproducibility

PacBio HiFi reads from two field isolates were assembled de novo with hifiasm and NextDenovo, and the NextDenovo assemblies were kept after QC. Assemblies were annotated for genes, repeats and effectors, compared with reference genomes, and complemented with a leaf-infection virulence assay.

  • PacBio HiFi long-read sequencing of isolate M22.7 (wild rice, Mondego basin), 585,514 reads, mean length 16,724 bp · Pyricularia oryzae · n = 1 isolate; 585,514 reads · BioProject PRJNA1451107; BioSample SAMN57182087; GenBank JBXBCC000000000
  • PacBio HiFi long-read sequencing of isolate T22.2 (cultivated rice var. Lusitano, Tejo basin), 423,346 reads, mean length 18,356 bp · Pyricularia oryzae · n = 1 isolate; 423,346 reads · BioSample SAMN57183957; GenBank JBXBCB000000000
  • Annotated mitochondrial genomes of both isolates · Pyricularia oryzae · n = 2 genomes · GenBank PZ276150 and PZ276151
  • Detached-leaf infection assays on rice cultivars Presto and Bomba · rice · n = Presto n = 15 and 16; Bomba n = 32 and 30 (two experiments)
  • Reference genome used for QUAST comparison (isolate 70-15) · Pyricularia oryzae · n = 1 assembly · GCA_000002495.2
Methods & tools
hifiasm, NextDenovo, BlobToolkit, BUSCO, EukCC, MitoHiFi, Inspector, QUAST, Funannotate, antiSMASH, EffectorP, RepeatMasker
Data & accessions
https://zenodo.org/records/19556489
Reagents & environment
Software versions are given in the text (e.g. hifiasm v0.25.0-r726, NextDenovo v2.5.2, BUSCO v5.8.3, Funannotate v1.8.17); no container or requirements file is mentioned.
Assay conditions & compute
NextDenovo was run with sort options set to 450 GB memory and 64 threads; no runtimes are reported.
Reproducibility gaps
No analysis scripts are provided. Phylogeny, dotplots, PCR and conidiation data are in supplementary material not included in the text. The isolates are held in the MUM fungal collection.
Limitations (5)
  • Mini-chromosomes were not assessed; this would need karyotype analysis by CHEF electrophoresis. · stated by the authors
  • The reduced conidiation of M22.7 was seen only in vitro and would have to be confirmed in planta before it can be read as a fitness disadvantage. · stated by the authors
  • The effector-count difference between isolates may just reflect the difference in total gene number; a pangenomic analysis with infection data is needed to test functional implications. · stated by the authors
  • A mitochondrial contig assigned to Penicillium was removed from M22.7, and the authors cannot exclude an isolated contamination in that culture. · stated by the authors
  • Only two isolates were sequenced, and virulence was tested only on detached leaves of two cultivars, so the Avr gene loss cannot be linked to broader host range or field behaviour. · noted in this summary

Who should read:Read this if you work on rice blast or P. oryzae genomics and want recent long-read European reference assemblies, annotations and an isolate lacking Avr-Pii, Avr-Pia and Avr-Pik.

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