Objective
- Collect Fusarium oxysporum f. sp. lini (Fol) isolates from across the flax growing areas of SK and MB.
- Characterize the pathogenicity of isolates through indoor phenotype screening.
- Sequence selected isolates with NGS and analyze genetic variance with association, pathogenicity, virulence and geological origins.
- Develop PCR assay(s) to differentiate isolates.
Project Description
Fusarium wilt of flax is a devastating soil-borne disease caused by Fusarium oxysporum f. sp. lini (Fol) that, without host resistance, can decimate the crop and it can be impossible to grow flax on that land for many years. Host resistance is the main defense against this disease. Understanding the genotypic components of the pathogenicity of Fol would be beneficial for development of flax lines with better resistance. The development of molecular markers could be used to differentiate pathogenic Fol from non-pathogenic F. oxysporum isolates without labourious screening. This project was conducted: i) to collect Fol isolates from the flax growing regions in SK and MB, ii) to characterize the pathogenicity of isolates, iii) to whole-genome sequence selected Fol isolates with NGS (Next Generation Sequencing) technologies and analyze genomes for association with pathogenicity and areas of origin, and iv) to develop PCR assay(s) to differentiate flax pathogenic isolates from non-pathogenic isolates. After sample collection in 2020 and 2021 in SK and MB, 79 isolates were identified as F. oxysporum by sequence comparison of the TEF-1α gene in each isolate. Seventeen isolates collected in SK and MB in 1997 and 1998 were also added to the collection and identified as F. oxysporum. All isolates were screened for pathogenicity and virulence level on four flax cultivars that varied in resistance to Fusarium wilt (‘AAC Bright’, ‘Bison’, ‘NorLin’ and ‘Novelty’). Based on the results of the pathogenicity phenotypes, 24 isolates were selected for their variable pathogenicity, virulence levels and geographical origin (SK or MB). DNA was extracted from each isolate and submitted for whole genome sequencing with PacBio HiFi sequencing technology and de novo assembly of the sequenced reads. The results included long-read sequences and contigs with high contiguity and completeness for 23 isolates. GWAS (Genome Wide Association Study) was performed with the sequence reads for pathogenicity to flax overall and pathogenicity to specific cultivars. A single SNP was identified to have a highly significance association with pathogenicity to flax and a few SNPs were identified to be associated with virulence to ‘NorLin’ (moderately resistant). BLAST search for Secreted In Xylem (SIX) genes (effector genes involved in fungal infection) in the contigs of each isolate revealed that all pathogenic Fol isolates from any location or of any virulence level possessed complete or partial copies of six families of SIX genes (SIX1, SIX7, SIX9, SIX10, SIX12, and SIX13) except for two non-pathogenic isolates from a wilt nursery in Saskatoon that possessed all SIX genes. The analyses identified potential SNPs and genes that can be used for designing primers to develop PCR assays to distinguish pathogenic Fol isolates from non-pathogenic isolates. Further genome analysis is needed to understand and identify genomic mechanisms for pathogenicity to flax.
Grower Benefits
- Developed an effective wilt disease screening method under controlled environment that can be used for pathogen identification or flax line screening for resistance.
- Created a collection of F. oxysporum isolates with information on origin, pathogenicity and virulence levels to flax.
- Obtained high quality whole genome sequences of F. oxysporum isolates with different pathogenicity and virulence phenotypes.
