Development of KASP Markers in Association with Avirulence Genes and Utilization of a CYP51 KASP Marker to Determine Dynamics of DMI Fungicide Sensitivity in the Stripe Rust Pathogen, Puccinia Striiformis

Hannah C Merrill

doi:10.7273/000007423

Puccinia striiformis f. sp. tritici (Pst), the causal organism of stripe rust of wheat, and Puccinia striiformis f. sp. hordei (Psh), the causal organism of stripe rust of barley, are important pathogens threatening wheat and barley production in the world, respectively. Stripe rust is controlled by developing and growing resistant cultivars, and/or applying fungicides when needed. However, the stripe rust fungus can develop virulence races circumventing race-specific resistance genes in the host plants and strains tolerant to fungicides. The objectives of this study were to 1) develop Kompetitive Allele Specific PCR (KASP) markers associated with avirulence genes in Pst and 2) determine the dynamics of the demethylation inhibitor (DMI) fungicide targeting gene CYP51 mutants in both Pst and Psh in the United States using a specific KASP marker. To develop markers associated with avirulence genes, primers for KASP markers were designed using previously identified secreted protein gene-based single nucleotide polymorphism (SP-SNP) markers associated with avirulence genes in Pst. A total of 92 KASP markers were designed and screened using a panel of 192 Pst isolates selected based on their race, multilocus genotype, country of origin, and year of collection. Twenty-three of the KASP markers met the criteria of sorting the Pst isolates into three genotype groups, XX, XY, and YY, and these markers were further tested with 845 Pst isolates collected from 2019 to 2021. Twenty-one of those markers were significantly associated with 16 avirulence genes, and most markers were applicable to two or more avirulence genes. Similarly, most avirulence genes had two or more useful markers. Sixteen markers used in different combinations with up to three markers in each combination could provide specific detection of the 16 avirulence genes. To determine the dynamics of DMI fungicide tolerance in the Pst and Psh populations in the United States, the KASP marker for the point mutation (from nucleotide A to T for the Y134F amino acid change) in the CYP51 gene was used to genotype 3,329 Pst isolates collected from 1968 to 2021 and 583 Psh isolates collected from 1993 to 2021. In addition, 190 Pst-infected leaf samples collected from different treatments of Tilt in field fungicide-testing plots were also genotyped using the KASP marker. The marker separated the isolates into the AA (wildtype), AT (heterozygous mutant), and TT (mutant) genotypes and determined frequencies of the three genotypes in each year, revealing the fluctuation of the mutant genotypes from year to year. The genotyping analysis of the fungicide testing leaf samples in 2024 supported the hypothesis that fungicide (Tilt) application increases mutant frequency, hence, increasing fungicide tolerance. Twenty-two isolates, including 16 Pst and 6 Psh isolates were tested for urediniospore germination at various concentrations of Tilt. Although at the full labeled rate, Tilt completely inhibited spore germination of the wild type and basically halted germ tube growth of the mutant types, significant increases of germination of the homozygous mutant were observed at the reduced concentration. Based on the germination data, the half maximal effective concentration (EC50) value of the homozygous mutant was more than twice the value of the wild type, proving that Tilt is less effective on the homozygous mutant than on the wild type. The KASP markers developed and used in this study should be useful in monitoring virulence genes and DMI fungicide tolerance in the stripe rust pathogen populations.

Development of KASP Markers in Association with Avirulence Genes and Utilization of a CYP51 KASP Marker to Determine Dynamics of DMI Fungicide Sensitivity in the Stripe Rust Pathogen, Puccinia Striiformis

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