Development of a diagnostic assays for race differentiation of Podophaera macularis

Authors: BLOCK, MARY - Oregon State University; KNAUS, BRIAN - Oregon State University; WISEMAN, MICHELE - Oregon State University; Grunwald, Niklaus - Nik; Gent, David - Dave

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/5/2020
Publication Date: 4/22/2021
Citation: Block, M., Knaus, B.J., Wiseman, M., Grunwald, N.J., Gent, D.H. 2021. Development of a diagnostic assays for race differentiation of Podophaera macularis. Plant Disease. 105(4):965–971. https://doi.org/10.1094/PDIS-06-20-1289-RE.
DOI: https://doi.org/10.1094/PDIS-06-20-1289-RE

Interpretive Summary: Powdery mildew is an important disease of hop. Management of the disease is complicated because multiple strains of the fungus exist that have varying ability to infect different cultivars. This means that when disease occurs, growers may not know with certainty which cultivars are at risk of disease. In this research, we identified genetic markers in the fungus that are associated with the ability of the pathogen to infect plants with the resistance gene termed R6. These genetic markers were then converted into a simple DNA-based diagnostic assay. The assay had perfect discrimination of strains of the fungus that can infect plants with disease resistance based on R6 versus those that cannot. However, the performance of the assay was limited to powdery mildew samples in the western U.S. and did not predict race of powdery mildew samples obtained from Europe. This assay has practical applications in breeding programs, research, and other settings where rapid confirmation of pathogen race is needed.

Technical Abstract: Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6-isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially-labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting multiplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak non-specific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, non-specification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.